Transgenerational toxicity of Aroclor 1232 by inhalational route in mouse model

Transgenerational toxicity of Aroclor 1232 by inhalational route in mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transgenerational toxicity of Aroclor 1232 by inhalational route in mouse model Sivaselvakumar Muthusamy, Ramanujam Narayanan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4820257/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Aroclor 1232 is a commercial mixture of polychlorinated biphenyls with occupational toxicological implications as it contains semi-volatile congeners like PCB 77, along with highly lipophillic ones like PCB 180. We aimed to assess the trans-generational behavioral toxicities of this complex mixture in mice by inhalational dosing chamber. We also aimed to evaluate the penetrability of PCBs across placental and lactational routes in mice in this study. Methods We assessed the probable route of penetration in the first generation of litters using behavioral scores as surrogate markers of developmental toxicity. We also quantified plasma concentrations of key PCBs and anthropometric parameters for trans-generational comparability. Results PCBs are responsible for behavioral toxicities across one generation of mice by occupational exposures via the inhalational route. Behavioral scores of F1 and F2generation mice indicated as surrogate endpoints that PCBs are concentrated in lactating milk more than placental route in the next generation of mice. No significant lethality or effects on anthropometrical parameters were detected across one generation although the congeners were detected in plasma of the litters when they were 12–14 weeks age. Conclusion PCBs may pose both indoor and outdoor occupational hazard by inhalational routes and cause behavioral deficits across one generation by transfer via lactational routes. PCB 77 is more evident of penetrating trans-generationally and produces behavioral toxicities in subsequent generations. They definitely carry inhalational hazard in workplaces and outdoor as environmental pollutants and neuroendocrine disruptors. Inhalational toxicity Polychlorinated Biphenyl (PCB) trans-generational toxicity occupational hazard assessment developmental toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Aroclor 1232 is a highly semi-volatile, lower chlorinated Polychlorinated Biphenyl (PCB) mixture that has been used in rubber industry as plasticizer and in electronic equipment for its thermal inertness. 1 It constitutes many congeners, PCB 77 and PCB 180, as key congeners of toxicological and pharmacokinetic significance. PCB 77 is a major dioxin-like PCB congener that exerts its toxicological effects by its affinity to the Aryl Hydrocarbon Receptor family. 2 PCB 180 is a highly persistent, heptachlorinated lipophilic congener that has a higher biomagnification factor. 3 Whereas PCB 77 has neuroendocrine disruptive toxicities, PCB 180 has mainly cardiometabolic toxicities. 4,5 PCBs are known to penetrate in-utero from environment and cause neurobehavioral toxicities. 6 However, their mode and route of persistence as well as the characterization of their developmental toxicities after in-utero and lactational penetration are less well evaluated. Inhalational route of PCB exposure is unique in the manner that it causes bioaccumulation of the congeners on repeated low dose exposures, 7 which may be enough to allow the congeners’ penetration into the placental and lactational routes across a generation, which we had hypothesized. This could theoretically lead to enough brain concentrations in the offsprings to cause neurobehavioral disruption. In this study, we aim to provide a proof-of-concept evidence to test our two-fold hypothesis, as stated above, using inhalational exposure of Swiss albino mice to 4 mg/cu.m of Aroclor 1232 in an inhalational chamber. We have chosen this dose based on dose calculations via a thorough animal dose extrapolation from acute to sub-acute exposures using benchmark approach. 8,9,10,11 We provide the evidence of subtle but biologically significant results of the mode of penetration of Aroclor 1232 via inhalational route across one generation, and the qualification of the type of neurodevelopmental toxicities that it causes. Materials and methods Chemicals Aroclor 1232 mixture (Supplemental Table 1) and the reference standard of PCB 77 and PCB 180 were purchased from Sigma-Aldrich, India. Acetonitrile and methanol of HPLC grade were purchased from Sigma-Aldrich, India. Milli-Q purified water was used from Merck-Millipore Inc., India. Source of Animals and Ethics approvals : The Sixteen Swiss Albino mice from parent generation, and the pups of F1 and F2 generations were obtained from the Institutional Animal Facility (PSG Animal Facility) with prior license for in-house breeding for small animal species and supply of animals for Research, which is recognized by the Indian regulator in animal experiments, the CPCSEA, after prior approval of the IAEC (PSG-IAEC proposal approval numbers: 207/2013/IAEC, 207/2014/IAEC and 207/2015/IAEC, each valid for one year; CPCSEA Animal Facility approval number 158/PO) with permission for in-house breeding from CPCSEA (158/PO/bc/99/CPCSEA and 158/PO/ReBi-SL/99/CPCSEA). The animals for BAL phase of the study were approved by IAEC also {IAEC approval number: 392/2018 and has utilized three Swiss albino mice for the conduct of this study phase} obtained from the CPCSEA-approved PSG Animal facility [CPCSEA Animal facility approval number: 158/PO/ReBi-S/Re-L/99/CPCSEA]. Animals : Sixteen Swiss albino female mice were randomized and acclimatized in separate cages (triplets per cage). Prior to acclimatization, these mice were grouped as control (G-I) and treated (G-II), eight mice each group. All the methods used for the trans-generation toxicity study were approved by the Institutional Animal Ethics Committee (IAEC), and were in lines with the CPCSEA guidelines for animal facility, 2005, 12 OECD guidance on acute toxicity studies: GD 39, 13 and AVMA guidelines of animal euthanasia, 2013. 14 Dose extrapolation - Acute single (6-hours / dose) inhalational exposure of 8 mice with 1 ppm, 2 ppm, and 4 ppm of Aroclor 1232 in the chamber was performed, and the lethality was used to model the benchmark dose (BMD) and BMD-limit-95%, for Aroclor 1232, used later to derive the minimum toxic dose of Aroclor 1232, for use in subacute two-week exposures in the trans-generation toxicity study. The EPA-BMD Software 10 was used for this purpose (BMDS version 2.0, US-EPA). Study Design - Exposure of Parent generation (P) The control mice (G-I) were treated with water aerosolization for six hours a day; five days a week for two weeks. The PCB- treated mice (G-II) were nebulized with Aroclor 1232 using with 4 mg/cu.m dose of the PCB mixture using the same dosing protocol. The hourly behavior scores were rated for both groups during the two-week treatment sessions. After two-week treatment, the Group I mice were rehabilitated back to the Animal Facility; whereas the Group II (N = 8) mice were mated with two other male mice per female mice (pair mating) obtained separately from the Animal Facility (Fig. 1 ). Study Design -Trans-generation toxicity study In Group-II after gestation confirmation, two mice were not pregnant (G-III) and six mice were pregnant (G-IV). The G-III mice were observed for delayed toxic signs till 12–14 weeks of age and G-IV pregnant mice were grouped into two cohorts of three mice each namely, G-IV-A; G-IV-B. The G-IV-A mice were exposed to the same dose and time protocol of Aroclor 1232 from days 1–14 after confirmation of pregnancy and G-IV-B was not exposed. After two weeks, G-IV-A and G-IV-B group of each one mouse delivered twenty pups (8 female and 12 male in each group) which comprised the first offspring (F1) generation of mice. The mice which did not deliver were cared for two more weeks and rehabilitated back. All pups which delivered in the Animal facility were also cared for general health, in the corresponding home cages along with their mothers, after the parent fathers were separated due to risk of cannibalism. The pups of the two female mice which delivered 20 litters each were assessed for lethality as an indication of teratogenicity indirectly. Five female pups (Group IV-A) and five female pups (Group IV-B) died due to prematurity on Post-Natal Day, PND 0. The rest of unused male pups (except the 3 female mice of Group IV-A and Group IV-B each) were rehabilitated back. In Group IV-A, three female mice pups were selected and named as Group V-F1 (G-V-F1) and then exposed to Aroclor1232 at a dose of 4 mg/m 3 for 2 weeks (Gestational Days, GD 0–13 days) for ascertaining the chance of transplacental penetrability of PCBs. On GD 15–17, they were assessed for behavior, anthropometry, and then observed till 12–14 weeks of age for delayed signs of toxicity and plasma harvested for estimating PCB 77 and PCB 180.Their pups were rehabilitated back at the Animal Facility. Likewise, pups of Group VI-F1 (N = 3 female mice) were allowed to mate with one male mice after reaching sexual maturity at 8 weeks age and deliver litters (Group VII-F2). Delivery was confirmed only in one (Group VI-F1A) of the three mice on GD 0 using yellow plug formation in the vaginal swab as an indication of gestation. This pregnant female delivered 3 male pups which were rehabilitated, and five female pups which formed GroupVII-F2 (N = 5 females, F2 generation). GroupVII-F2 mice were exposed to Aroclor 1232 at a dose of 4 mg/m 3 for 2 weeks (PND 7–21 days) for ascertaining the chance of lactational penetrability of PCBs. On PND 24–27, they were assessed for behavior, development, anthropometry, and then observed till 12–14 weeks of age for delayed signs of toxicity and plasma harvested for estimating PCB 77 and PCB 180.All mice were then rehabilitated back at the Animal Facility. Outcome measurements: Hourly behavioral assessments using Irwin scale was undertaken for all the six groups during the two-weekly exposures, for comparison and penetrability evaluations between Groups I, II, V-A, and VII. Anthropometric measurements were compared between groups V-A and VII. The amount of PCB 77 and PCB 180 in plasma samples of experimental mice was estimated using UPLC-DAD by previously reported method and compared with P-generation (Group II), F1 generation (Group V-A) and F2 generation (Group VII). 15 All exposures were given in a whole-body inhalational chamber, with periodical monitoring of chamber environment. BAL study - lung histopathology: The authors’ methodology 16 was applied for sampling, sectioning and staining of lung sections (Supplementary Table 2) as per regulatory requirements. 17 Briefly, three animals were selected and weighed. BAL ringer fluid solution (1x PPB saline in dd.H 2 O at 4 ⁰C) was prepared. Methanol solution was prepared as negative control. One animal was preselected as positive control animal. The second animal was given 1 ppm methanol inhalation in an inhalational chamber. The third mouse was given 1 ppm Aroclor 1232 in a whole body inhalational chamber. Under Ketamine euthanasia IV at 55 mg/kg with Xylazine 1 mg/kg IV at least fifteen minutes prior to the BAL technique, after tracheostomy was secured (40 n white thread used as probe for carina), the BAL ringer was perfused into the mouse lungs using 21 G tube inserted to a balloon catheter thrice. The aspirated BAL-fluid was incorporated onto 1 ml tubes with PBS prefilled at three times the volume of the BAL fluid yield, centrifuged at 300 g for 5 minutes at 4 C and analyzed for cytology, Aroclor concentrations using UPLC-DAD and inflammatory markers. Lung tissue can be additionally harvested for assessing inflammation using H&E staining. Data availability The key datasets generated for the study can be found in the Research Square at: { https://doi.org/10.21203/rs.3.rs-1425455/v1 }. Additional datasets can be provided on request. Results The trans-generation subacute toxicity study was conducted with occupational inhalational exposures of mice to 4 mg/cu.m of Aroclor 1232, and the penetrability of this mixture was ascertained along with the elucidation of its effects on the mice behavioral scores. All the environmental parameters of mice in the whole-body inhalational chamber were within acceptable, tolerable limits, as per CPCSEA and OECD guidelines. 12, 17 Chamber environment: Relative humidity was 30–80%, temperature was 37–37 0 C, pressure was 731–739 mm Hg, oxygen was 21%, and aerosol flow rates were 16–18 L/min in the chamber throughout the exposures (Supplemental Fig. 1). Bronchoalveolar Lavage (BAL) cytology and histopathology : The trachea of mice exposed to the highest dose level (16 mg/ cu.m) exhibited sheets of lymphocytes with dense inflammation, as compared to control animals [Figure 2 A] on H&E staining of BAL tissue [Figure 2 B] with presence of mast cells, alveolar edema, and lung parenchymal eosinophilic infiltrates [Figure 2 C]. Minimal inflammation was observed for all the three mouse lung samples. BAL-fluid cytology showed Lymphocytosis and granulocytosis in Aroclor treated mouse as compared to both controls. Benchmark dose : A Benchmark Dose (BMD) of 1 mg/cu.m was extrapolated from acute toxicity study of 1 ppb of Aroclor 1232. Using this approach and as per PEL values based on acceptable regulatory values of PCBs indoor, the dose for subacute study was fixed at 4 mg/cu.m [Figure 3 ]. Anthropometric and behavioral assessments : The tail to body trunk ratio of all mice did not differ significantly [2:1 to 4:1, p > 0.05] between groups V-A and VII (Supplementary Table 3). The median body weight of pups (Group V-A and VII) on day 7 were 3.75–7.30 grams. The total and mean behavioral scores was reduced significantly in the group VII compared to parent control group I [3224 ± 6.61; versus, 4283 ± 5.81; Fig. 4 ]. A two-way ANOVA across all individual pairs of data in all the four groups–groups I, II, V- A and VII in pups was performed to determine the probable route of PCB transfer across one generation. The group VII behavioral scores were significantly different on most of the ten days of exposure compared to all the three other groups, pair wise comparison yielding p values from < 0.001 to < 0.05 on most days. However, none of the other pair-wise comparisons resulted in statistical significance across any days of exposure. The mean scores were significantly lower in group V compared to all the other groups at all 10 days except day 5 and 8 of exposure; however, no such significant difference existed between Groups I and V-A mice in the behavior scores (Fig. 4 , p < 0.05). Also, the group x hours of exposure variances contributed significantly to the difference in scores across days of exposure as indicated by post hoc comparisons (p < 0.05). The concentrations of the two prototypical compounds in Aroclor 1232 mixture, PCB 77 and PCB 180 were below Lower Limit of Quantification (LLOQ) in group VI. The mean plasma concentration of PCB 77 was 88.32 ± 2.12 ng/mL, and those of PCB 180 in plasma was 66.49 ± 1.76 ng/mL in Group II mice. As compared to Peripheral Nervous system scores, the domains that differed significantly across Groups II, V-A and VII were Autonomic Nervous system followed by Central Nervous functions (Figs. 5 , 6 , Supplementary Table 4). Depression in all domain behavioral scores was maximum in the Group VII as compared to other two groups. Discussion A two-generational inhalational toxicity risk assessment was conducted in mice using an inhalational chamber and Aroclor 1232 to ascertain the route of trans-generational transfer of PCBs and neurobehavioural toxicities in F1-F2 generation after dosing in P generation at geometrically progressive dose levels. A definite depression of behavior in F1 generation pups was seen compared to parent generation owing to higher transfer of PCBs across lactational route, as concluded in earlier studies. 18 From the study, there does not seem to be any transfer of PCBs through placenta probably due to their higher lipophilicity. This agrees with earlier studies that PCBs tend to persist more in milk than other body fluids in rodents and cause neurodevelopmental effects in pups of exposed mothers. 19 The effect of such short duration of exposure on PCB 180 transfer by other dosing routes across one generation has also been discussed earlier by other studies in rats. 20 In addition, PCB 77 diminished normal adult sexual partner preference in rats treated during gestation and post-natal period orally, though it did not affect sexual behavior, and this effect dependent whether the exposure was in-utero or lactational, and was independent of any other behavior phenotypes. 21 PCB 77 exposure during pre-natal period in maternal rodents altered maternal behavior including increased maternal nursing, and amount of maternal auto grooming. Increased time spent in nest, increased pup grooming frequencies, reduction in high crouch nursing and pup-maternal interactions were both due to maternal in-utero and post-natal exposure to PCB 77. 22 Such changes were also ascribed to altered preference to maternal odor although the preference to novel odors did not change, after exposure to PCB 77, a process called ‘odor conditioning behavior.’ 23 PCB 77, the most toxic DL PCB can reduce Thyroxine both peripherally and centrally in key stages of neurodevelopmental differentiation in chicken, whereas, more abundant o-substituted NDL- PCBs like PCB 153 and 180 did not affect thyroid hormones – a key mechanism for neuroendocrine disruption caused by PCBs developmentally. 24 Both PCB 180 and PCB 77 caused increased mobilization of hepatic retinoid stores with increased renal retinoid acid levels with reduced thyroid hormone levels; this is of key importance in neurodevelopmental processes since retinoic acid derivatives are lipid derived antioxidants. 25 Although the parent mice brains showed the presence of PCB 77 and PCB 180 in low concentrations, the concentrations of both compounds were below the LLOQ in the offspring mice at 14 weeks of age – probably due to low analytical detectability and also due to less cumulation due to shorter duration of exposure of 2 weeks, as showed by the lower peak brain concentration (Cp) at 48 hours in the parent generation mice. The effect of such short duration of exposure on PCB 180 transfer by other dosing routes across one generation has also been discussed earlier by other studies in rats and humans. 26,27 In summary, the study results prove the existence of hazardous plausibility of neurotoxicological risk of PCB exposures across at least two generations after inhalation exposures. These risks could get amplified in human workers both via indoor (occupational) and environmental exposures and be passed on to next generations translating onto behavioral, neurological and cognitive imbalances and disorders. Such deficits have been assessed in human participants indoor and after environmental exposures also. 28,29,30,31 The probable route of trans-generational transfer of PCBs would be lactational one as compared to the placental route. These changes in anthropometric outcomes (Supplementary Table 3) correlate well with alterations in brain Noradrenaline (Fig. 7 ) and behavior scores in social activity (Supplementary Table 4, Fig. 8 ) over 14 day exposures, with an additional 7-day observation period. This pilot study provides proof-of-concept evidence on the causality of neurobehavioural deficits in mice as caused by occupational exposures of parent mice via inhalational routes at a dose of 4 mg/cu m. Moreover, this could be due to transfer of PCB congeners across a generation by lactational concentrations due to their basic nature and higher lipophilicities. Although the P generation sample size was lower than the recommended size for statistical significance, the study outcomes are biologically significant and as per the standard CPCSEA guidelines for animal housing, which were also applied to housing inside the inhalational chamber, the sample size of parents were kept minimal for ensuring animal welfare. This pivotal study yields valid and robust results of the epigenetic transgenerational theory of endocrine disruptors as etiological factors in disease pathophysiology in rodents, for confirmation in further studies. Abbreviations AhR = Aryl Hydrocarbon receptor; AVMA = American Veterinary Medical Association; ANOVA = Analysis of Variance; BAL = Bronchoalveolar Lavage; BMD = Bench Mark Dose; CPCSEA = Committee for the Purpose of Control and Supervision of Experiments on Animals; EPA = Environment Protection Agency; GD = Gestational Day; H&E = Haematoxylin & Eosin; HPLC = High Performance Liquid Chromatography; IAEC = Institutional Animal Ethics Committee; LLOQ = Lower Limit of Quantification; OECD = Organization for Economic Cooperation and Development; PCB = Polychlorinated Biphenyl; PEL = Permissible Exposure Limit; PND = Postnatal Day; UPLC-DAD = Ultra-High Performance Liquid Chromatography-Diode Array Detector Declarations Acknowledgements The author would also like to acknowledge the facilities given by PSG Institute of Medical Sciences & Research, Coimbatore for the success of the work. The author would like to acknowledge Dr. Ramalingam Sankaran (**deceased), former Dean and Professor of Pharmacology, for his untiring efforts in provision of essential facilities and administrative guidance in the PSG Center for Molecular Medicine & Therapeutics, useful in the execution of the work undertaken in the laboratory. The author acknowledges his role as the Guide for PhD work, of which this work is a key component. The authors like to acknowledge Dr S. Vidhiyalakshmi, Faculty in the department of Pathology, for her help and assistance rendered in staining of lung sections after BAL study sampling. Funding The authors state that the study was self-funded by the authors, and declare that no other sources of extramural funding exist. Ethics & Regulatory Declaration All methods were followed in this animal study after prior IAEC approval in compliance with the CPCSEA guidelines (2005), AVMA guidelines (2013) and OECD guidance document GD 39. Competing interests The authors declare no conflicts of interests during the conception and inception of the paper, including any non-financial and monetary ones. Supplementary material Supplementary material (Supplementary Tables 1-4) is available at Brain online’. References Agency for Toxic Substances and Disease Registry, Toxicological profile for polychlorinated biphenyls (PCBs), November 2000, accessed at https://www.atsdr.cdc.gov/ToxProfiles/tp17.pdf. Erin N. Jackson,Sean E. Thatcher,NikaLarian,VictoriaEnglish,SonySoman,Andrew J. Morris,JiayingWeng,ArnoldStromberg,Hollie I. Swanson,KevinPearson,and Lisa A. Cassis, 2019, Effects of Aryl Hydrocarbon Receptor Deficiency on PCB-77-Induced Impairment of Glucose Homeostasis during Weight Loss in Male and Female Obese Mice; Environmental Health Perspectives 127:7 CID: 077004 https://doi.org/10.1289/EHP4133. 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Cognitive development of Yu-Cheng ('Oil Disease') children prenatally exposed to heat-degraded PCBs. Jama. 1992 Dec 9;268(22):3213-8. Holmes AK, Rubin C, Marcus M, Kieszak S, Jones RW, Golding J, McGeehin M. A pilot study of chemical exposures among mothers and children enrolled in the Avon Longitudinal Study of Parents and Children. InAnnual Meeting. Marek RF, Thorne PS, Wang K, DeWall J, Hornbuckle KC. PCBs and OH-PCBs in serum from children and mothers in urban and rural US communities. Environmental science & technology. 2013 Apr 2;47(7):3353-61. Supplementary Figure 1 Supplementary Figure 1 is not available with this version Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4820257","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336796083,"identity":"9a8e2f90-efc9-467f-8834-690a1892bb39","order_by":0,"name":"Sivaselvakumar Muthusamy","email":"","orcid":"","institution":"PSG Institute of Medical Sciences \u0026 Research Peelamedu","correspondingAuthor":false,"prefix":"","firstName":"Sivaselvakumar","middleName":"","lastName":"Muthusamy","suffix":""},{"id":336796084,"identity":"3ced141e-6cc8-4e0f-81b6-13b5add0fa07","order_by":1,"name":"Ramanujam Narayanan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYLCChAoJOX7+5gNApoQMcVoenLEwlpxxLAGkhYcoHYwP2yoSNxzIMQBxCGsxbz/8dENim0TizIYzn1/dqLHgYWA/fHQDPi0yZ9LMbiSckzDuZ+7dZp1zDOgwnrS0G/i0SDAkALWUScjObDi7zTiHDahFgscMvxb+599uJLBJMAL98sw45x8xWiRygLa0SSgCtTA/zm0jSsubshsJZyRAgWzGnNsnwcNG0C/86dtu/qioA0Xl488534AM9sPH8GpBBmwSYJJY5SDA/IEU1aNgFIyCUTByAAARM0ynqB+bcwAAAABJRU5ErkJggg==","orcid":"","institution":"PSG Institute of Medical Sciences \u0026 Research Peelamedu","correspondingAuthor":true,"prefix":"","firstName":"Ramanujam","middleName":"","lastName":"Narayanan","suffix":""}],"badges":[],"createdAt":"2024-07-29 08:24:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4820257/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4820257/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64249330,"identity":"a4c924f9-ca7c-44f8-a83c-f7b7c17de1d9","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136895,"visible":true,"origin":"","legend":"\u003cp\u003eAnimal grouping for the trans-generational toxicity study of Aroclor 1232 by inhalational route.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/aae2fec27c2fdf3a58177172.jpg"},{"id":64249338,"identity":"9e9ea970-3277-42e2-9864-9d5eda26ff72","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1667675,"visible":true,"origin":"","legend":"\u003cp\u003eBAL Cytology showing control mouse respiratory tract (A), and three lungs of mice exposed to highest dose of Aroclor 1232 via inhalational route with inflammatory findings (B,C,D).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/799dde5a581467b1377c7a76.jpg"},{"id":64249335,"identity":"e274c862-f71a-4b28-858c-1a05eb879f8b","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43772,"visible":true,"origin":"","legend":"\u003cp\u003eDose extrapolation using US-EPA BMD [10] Software with Benchmark dose approach from acute toxicity study results using behavior scores after single 4 hour exposure of 1 ppb of Aroclor 1232.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/7ae5175bb7ea9db8f096c3ad.jpg"},{"id":64249473,"identity":"807cfe1b-70cc-4b39-8389-205e0185815f","added_by":"auto","created_at":"2024-09-10 21:09:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":299328,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 4mg.m\u003csup\u003e-3 \u003c/sup\u003eof inhalational Aroclor 1232 on mouse behavior across one generation.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/d1e8e5a149f9b9edc60e4363.jpg"},{"id":64249332,"identity":"bf5da6fd-b526-4ed3-b797-5d233b571dd6","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276339,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of hour of exposure on behavior scores in parent generation (Groups I \u0026amp; II). Contribution of time in this graph shows cumulative effect of Aroclor 1232 treatment over days are more important than hours of daily treatment.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/fd5f875df3ce9571a1c7122b.jpg"},{"id":64249687,"identity":"a315ed62-cf47-4f9e-a552-257913d9dbbf","added_by":"auto","created_at":"2024-09-10 21:17:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":258458,"visible":true,"origin":"","legend":"\u003cp\u003eFigure depicting domain subscores across two generations of mice after exposure to Aroclor 1232.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/a42971cc825ce5872b7ce490.jpg"},{"id":64249333,"identity":"d4cabb02-569e-407a-b6b9-4acd9c79cb98","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":51105,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in Noradrenaline at Tmax of PCB77 in brain.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/ad2621781009dd45525cef92.jpg"},{"id":64249337,"identity":"b4232785-d388-4b2c-95c4-823b60861995","added_by":"auto","created_at":"2024-09-10 21:01:46","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":49229,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in social activity of mice exposed to Aroclor 1232 for 2 week period in offspring generations.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/e355256dcfdd7f7cc07b5a7a.jpg"},{"id":76281803,"identity":"c900a501-a7c2-46fb-8b5d-7ce5d8ceda16","added_by":"auto","created_at":"2025-02-14 10:39:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3362796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/8213591b-184a-46a6-b2b1-48b73cdf295c.pdf"},{"id":64249472,"identity":"b9b85b8f-cf19-409f-a341-b5f5934c303c","added_by":"auto","created_at":"2024-09-10 21:09:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22624,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4820257/v1/88db7679773b3ff9b07e74d5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transgenerational toxicity of Aroclor 1232 by inhalational route in mouse model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAroclor 1232 is a highly semi-volatile, lower chlorinated Polychlorinated Biphenyl (PCB) mixture that has been used in rubber industry as plasticizer and in electronic equipment for its thermal inertness.\u003csup\u003e1\u003c/sup\u003e It constitutes many congeners, PCB 77 and PCB 180, as key congeners of toxicological and pharmacokinetic significance. PCB 77 is a major dioxin-like PCB congener that exerts its toxicological effects by its affinity to the Aryl Hydrocarbon Receptor family. \u003csup\u003e2\u003c/sup\u003e PCB 180 is a highly persistent, heptachlorinated lipophilic congener that has a higher biomagnification factor. \u003csup\u003e3\u003c/sup\u003e Whereas PCB 77 has neuroendocrine disruptive toxicities, PCB 180 has mainly cardiometabolic toxicities. \u003csup\u003e4,5\u003c/sup\u003e PCBs are known to penetrate in-utero from environment and cause neurobehavioral toxicities. \u003csup\u003e6\u003c/sup\u003e However, their mode and route of persistence as well as the characterization of their developmental toxicities after in-utero and lactational penetration are less well evaluated.\u003c/p\u003e \u003cp\u003eInhalational route of PCB exposure is unique in the manner that it causes bioaccumulation of the congeners on repeated low dose exposures, \u003csup\u003e7\u003c/sup\u003e which may be enough to allow the congeners\u0026rsquo; penetration into the placental and lactational routes across a generation, which we had hypothesized. This could theoretically lead to enough brain concentrations in the offsprings to cause neurobehavioral disruption.\u003c/p\u003e \u003cp\u003eIn this study, we aim to provide a proof-of-concept evidence to test our two-fold hypothesis, as stated above, using inhalational exposure of Swiss albino mice to 4 mg/cu.m of Aroclor 1232 in an inhalational chamber. We have chosen this dose based on dose calculations via a thorough animal dose extrapolation from acute to sub-acute exposures using benchmark approach. \u003csup\u003e8,9,10,11\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWe provide the evidence of subtle but biologically significant results of the mode of penetration of Aroclor 1232 via inhalational route across one generation, and the qualification of the type of neurodevelopmental toxicities that it causes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cstrong\u003eChemicals\u003c/strong\u003e \u003cp\u003eAroclor 1232 mixture (Supplemental Table\u0026nbsp;1) and the reference standard of PCB 77 and PCB 180 were purchased from Sigma-Aldrich, India. Acetonitrile and methanol of HPLC grade were purchased from Sigma-Aldrich, India. Milli-Q purified water was used from Merck-Millipore Inc., India.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eSource of Animals and Ethics approvals\u003c/b\u003e: The Sixteen Swiss Albino mice from parent generation, and the pups of F1 and F2 generations were obtained from the Institutional Animal Facility (PSG Animal Facility) with prior license for in-house breeding for small animal species and supply of animals for Research, which is recognized by the Indian regulator in animal experiments, the CPCSEA, after prior approval of the IAEC (PSG-IAEC proposal approval numbers: 207/2013/IAEC, 207/2014/IAEC and 207/2015/IAEC, each valid for one year; CPCSEA Animal Facility approval number 158/PO) with permission for in-house breeding from CPCSEA (158/PO/bc/99/CPCSEA and 158/PO/ReBi-SL/99/CPCSEA). The animals for BAL phase of the study were approved by IAEC also {IAEC approval number: 392/2018 and has utilized three Swiss albino mice for the conduct of this study phase} obtained from the CPCSEA-approved PSG Animal facility [CPCSEA Animal facility approval number: 158/PO/ReBi-S/Re-L/99/CPCSEA].\u003c/p\u003e \u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e: Sixteen Swiss albino female mice were randomized and acclimatized in separate cages (triplets per cage). Prior to acclimatization, these mice were grouped as control (G-I) and treated (G-II), eight mice each group. All the methods used for the trans-generation toxicity study were approved by the Institutional Animal Ethics Committee (IAEC), and were in lines with the CPCSEA guidelines for animal facility, 2005, \u003csup\u003e12\u003c/sup\u003e OECD guidance on acute toxicity studies: GD 39, \u003csup\u003e13\u003c/sup\u003e and AVMA guidelines of animal euthanasia, 2013. \u003csup\u003e14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDose extrapolation -\u003c/b\u003e Acute single (6-hours / dose) inhalational exposure of 8 mice with 1 ppm, 2 ppm, and 4 ppm of Aroclor 1232 in the chamber was performed, and the lethality was used to model the benchmark dose (BMD) and BMD-limit-95%, for Aroclor 1232, used later to derive the minimum toxic dose of Aroclor 1232, for use in subacute two-week exposures in the trans-generation toxicity study. The EPA-BMD Software \u003csup\u003e10\u003c/sup\u003e was used for this purpose (BMDS version 2.0, US-EPA).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStudy Design - Exposure of Parent generation (P)\u003c/strong\u003e \u003cp\u003eThe control mice (G-I) were treated with water aerosolization for six hours a day; five days a week for two weeks. The PCB- treated mice (G-II) were nebulized with Aroclor 1232 using with 4 mg/cu.m dose of the PCB mixture using the same dosing protocol. The hourly behavior scores were rated for both groups during the two-week treatment sessions. After two-week treatment, the Group I mice were rehabilitated back to the Animal Facility; whereas the Group II (N\u0026thinsp;=\u0026thinsp;8) mice were mated with two other male mice per female mice (pair mating) obtained separately from the Animal Facility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStudy Design -Trans-generation toxicity study\u003c/strong\u003e \u003cp\u003eIn Group-II after gestation confirmation, two mice were not pregnant (G-III) and six mice were pregnant (G-IV). The G-III mice were observed for delayed toxic signs till 12\u0026ndash;14 weeks of age and G-IV pregnant mice were grouped into two cohorts of three mice each namely, G-IV-A; G-IV-B. The G-IV-A mice were exposed to the same dose and time protocol of Aroclor 1232 from days 1\u0026ndash;14 after confirmation of pregnancy and G-IV-B was not exposed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eAfter two weeks, G-IV-A and G-IV-B group of each one mouse delivered twenty pups (8 female and 12 male in each group) which comprised the first offspring (F1) generation of mice. The mice which did not deliver were cared for two more weeks and rehabilitated back. All pups which delivered in the Animal facility were also cared for general health, in the corresponding home cages along with their mothers, after the parent fathers were separated due to risk of cannibalism. The pups of the two female mice which delivered 20 litters each were assessed for lethality as an indication of teratogenicity indirectly. Five female pups (Group IV-A) and five female pups (Group IV-B) died due to prematurity on Post-Natal Day, PND 0. The rest of unused male pups (except the 3 female mice of Group IV-A and Group IV-B each) were rehabilitated back.\u003c/p\u003e \u003cp\u003eIn Group IV-A, three female mice pups were selected and named as Group V-F1 (G-V-F1) and then exposed to Aroclor1232 at a dose of 4 mg/m\u003csup\u003e3\u003c/sup\u003e for 2 weeks (Gestational Days, GD 0\u0026ndash;13 days) for ascertaining the chance of transplacental penetrability of PCBs. On GD 15\u0026ndash;17, they were assessed for behavior, anthropometry, and then observed till 12\u0026ndash;14 weeks of age for delayed signs of toxicity and plasma harvested for estimating PCB 77 and PCB 180.Their pups were rehabilitated back at the Animal Facility.\u003c/p\u003e \u003cp\u003eLikewise, pups of Group VI-F1 (N\u0026thinsp;=\u0026thinsp;3 female mice) were allowed to mate with one male mice after reaching sexual maturity at 8 weeks age and deliver litters (Group VII-F2). Delivery was confirmed only in one (Group VI-F1A) of the three mice on GD 0 using yellow plug formation in the vaginal swab as an indication of gestation.\u003c/p\u003e \u003cp\u003eThis pregnant female delivered 3 male pups which were rehabilitated, and five female pups which formed GroupVII-F2 (N\u0026thinsp;=\u0026thinsp;5 females, F2 generation). GroupVII-F2 mice were exposed to Aroclor 1232 at a dose of 4 mg/m\u003csup\u003e3\u003c/sup\u003e for 2 weeks (PND 7\u0026ndash;21 days) for ascertaining the chance of lactational penetrability of PCBs. On PND 24\u0026ndash;27, they were assessed for behavior, development, anthropometry, and then observed till 12\u0026ndash;14 weeks of age for delayed signs of toxicity and plasma harvested for estimating PCB 77 and PCB 180.All mice were then rehabilitated back at the Animal Facility.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOutcome measurements:\u003c/h2\u003e \u003cp\u003eHourly behavioral assessments using Irwin scale was undertaken for all the six groups during the two-weekly exposures, for comparison and penetrability evaluations between Groups I, II, V-A, and VII. Anthropometric measurements were compared between groups V-A and VII. The amount of PCB 77 and PCB 180 in plasma samples of experimental mice was estimated using UPLC-DAD by previously reported method and compared with P-generation (Group II), F1 generation (Group V-A) and F2 generation (Group VII). \u003csup\u003e15\u003c/sup\u003e All exposures were given in a whole-body inhalational chamber, with periodical monitoring of chamber environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBAL study - lung histopathology:\u003c/h2\u003e \u003cp\u003eThe authors\u0026rsquo; methodology \u003csup\u003e16\u003c/sup\u003e was applied for sampling, sectioning and staining of lung sections (Supplementary Table\u0026nbsp;2) as per regulatory requirements. \u003csup\u003e17\u003c/sup\u003e Briefly, three animals were selected and weighed. BAL ringer fluid solution (1x PPB saline in dd.H\u003csub\u003e2\u003c/sub\u003eO at 4 ⁰C) was prepared. Methanol solution was prepared as negative control. One animal was preselected as positive control animal. The second animal was given 1 ppm methanol inhalation in an inhalational chamber. The third mouse was given 1 ppm Aroclor 1232 in a whole body inhalational chamber. Under Ketamine euthanasia IV at 55 mg/kg with Xylazine 1 mg/kg IV at least fifteen minutes prior to the BAL technique, after tracheostomy was secured (40 n white thread used as probe for carina), the BAL ringer was perfused into the mouse lungs using 21 G tube inserted to a balloon catheter thrice. The aspirated BAL-fluid was incorporated onto 1 ml tubes with PBS prefilled at three times the volume of the BAL fluid yield, centrifuged at 300 g for 5 minutes at 4 C and analyzed for cytology, Aroclor concentrations using UPLC-DAD and inflammatory markers. Lung tissue can be additionally harvested for assessing inflammation using H\u0026amp;E staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe key datasets generated for the study can be found in the Research Square at: {\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21203/rs.3.rs-1425455/v1\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-1425455/v1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e}. Additional datasets can be provided on request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe trans-generation subacute toxicity study was conducted with occupational inhalational exposures of mice to 4 mg/cu.m of Aroclor 1232, and the penetrability of this mixture was ascertained along with the elucidation of its effects on the mice behavioral scores. All the environmental parameters of mice in the whole-body inhalational chamber were within acceptable, tolerable limits, as per CPCSEA and OECD guidelines. \u003csup\u003e12, 17\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eChamber environment:\u003c/h2\u003e \u003cp\u003e \u003cem\u003eRelative humidity\u003c/em\u003e was 30\u0026ndash;80%, \u003cem\u003etemperature\u003c/em\u003e was 37\u0026ndash;37 \u003csup\u003e0\u003c/sup\u003eC, \u003cem\u003epressure\u003c/em\u003e was 731\u0026ndash;739 mm Hg, \u003cem\u003eoxygen\u003c/em\u003e was 21%, and \u003cem\u003eaerosol flow rates\u003c/em\u003e were 16\u0026ndash;18 L/min in the chamber throughout the exposures (Supplemental Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBronchoalveolar Lavage (BAL) cytology and histopathology\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe trachea of mice exposed to the highest dose level (16 mg/ cu.m) exhibited sheets of lymphocytes with dense inflammation, as compared to control animals [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA] on H\u0026amp;E staining of BAL tissue [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB] with presence of mast cells, alveolar edema, and lung parenchymal eosinophilic infiltrates [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC]. Minimal inflammation was observed for all the three mouse lung samples. BAL-fluid cytology showed Lymphocytosis and granulocytosis in Aroclor treated mouse as compared to both controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBenchmark dose\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eA \u003cem\u003eBenchmark Dose (BMD)\u003c/em\u003e of 1 mg/cu.m was extrapolated from acute toxicity study of 1 ppb of Aroclor 1232. Using this approach and as per PEL values based on acceptable regulatory values of PCBs indoor, the dose for subacute study was fixed at 4 mg/cu.m [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eAnthropometric and behavioral assessments\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003etail to body trunk ratio\u003c/em\u003e of all mice did not differ significantly [2:1 to 4:1, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05] between groups V-A and VII (Supplementary Table\u0026nbsp;3). The \u003cem\u003emedian body weight\u003c/em\u003e of pups (Group V-A and VII) on day 7 were 3.75\u0026ndash;7.30 grams. The \u003cem\u003etotal and mean behavioral scores\u003c/em\u003e was reduced significantly in the group VII compared to parent control group I [3224\u0026thinsp;\u0026plusmn;\u0026thinsp;6.61; versus, 4283\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA two-way ANOVA across all individual pairs of data in all the four groups\u0026ndash;groups I, II, V- A and VII in pups was performed to determine the probable route of PCB transfer across one generation. The group VII behavioral scores were significantly different on most of the ten days of exposure compared to all the three other groups, pair wise comparison yielding p values from \u0026lt;\u0026thinsp;0.001 to \u0026lt;\u0026thinsp;0.05 on most days. However, none of the other pair-wise comparisons resulted in statistical significance across any days of exposure. The mean scores were significantly lower in group V compared to all the other groups at all 10 days except day 5 and 8 of exposure; however, no such significant difference existed between Groups I and V-A mice in the behavior scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also, the group x hours of exposure variances contributed significantly to the difference in scores across days of exposure as indicated by post hoc comparisons (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003econcentrations\u003c/em\u003e of the two prototypical compounds in Aroclor 1232 mixture, PCB 77 and PCB 180 were below Lower Limit of Quantification (LLOQ) in group VI. The mean plasma concentration of PCB 77 was 88.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12 ng/mL, and those of PCB 180 in plasma was 66.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 ng/mL in Group II mice.\u003c/p\u003e \u003cp\u003e As compared to Peripheral Nervous system scores, the domains that differed significantly across Groups II, V-A and VII were Autonomic Nervous system followed by Central Nervous functions (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary Table\u0026nbsp;4). Depression in all domain behavioral scores was maximum in the Group VII as compared to other two groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA two-generational inhalational toxicity risk assessment was conducted in mice using an inhalational chamber and Aroclor 1232 to ascertain the route of trans-generational transfer of PCBs and neurobehavioural toxicities in F1-F2 generation after dosing in P generation at geometrically progressive dose levels.\u003c/p\u003e \u003cp\u003eA definite depression of behavior in F1 generation pups was seen compared to parent generation owing to higher transfer of PCBs across lactational route, as concluded in earlier studies. \u003csup\u003e18\u003c/sup\u003e From the study, there does not seem to be any transfer of PCBs through placenta probably due to their higher lipophilicity. This agrees with earlier studies that PCBs tend to persist more in milk than other body fluids in rodents and cause neurodevelopmental effects in pups of exposed mothers. \u003csup\u003e19\u003c/sup\u003e The effect of such short duration of exposure on PCB 180 transfer by other dosing routes across one generation has also been discussed earlier by other studies in rats. \u003csup\u003e20\u003c/sup\u003e In addition, PCB 77 diminished normal adult sexual partner preference in rats treated during gestation and post-natal period orally, though it did not affect sexual behavior, and this effect dependent whether the exposure was in-utero or lactational, and was independent of any other behavior phenotypes. \u003csup\u003e21\u003c/sup\u003e PCB 77 exposure during pre-natal period in maternal rodents altered maternal behavior including increased maternal nursing, and amount of maternal auto grooming. Increased time spent in nest, increased pup grooming frequencies, reduction in high crouch nursing and pup-maternal interactions were both due to maternal in-utero and post-natal exposure to PCB 77. \u003csup\u003e22\u003c/sup\u003e Such changes were also ascribed to altered preference to maternal odor although the preference to novel odors did not change, after exposure to PCB 77, a process called \u0026lsquo;odor conditioning behavior.\u0026rsquo; \u003csup\u003e23\u003c/sup\u003e PCB 77, the most toxic DL PCB can reduce Thyroxine both peripherally and centrally in key stages of neurodevelopmental differentiation in chicken, whereas, more abundant o-substituted NDL- PCBs like PCB 153 and 180 did not affect thyroid hormones \u0026ndash; a key mechanism for neuroendocrine disruption caused by PCBs developmentally.\u003csup\u003e24\u003c/sup\u003e Both PCB 180 and PCB 77 caused increased mobilization of hepatic retinoid stores with increased renal retinoid acid levels with reduced thyroid hormone levels; this is of key importance in neurodevelopmental processes since retinoic acid derivatives are lipid derived antioxidants. \u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough the parent mice brains showed the presence of PCB 77 and PCB 180 in low concentrations, the concentrations of both compounds were below the LLOQ in the offspring mice at 14 weeks of age \u0026ndash; probably due to low analytical detectability and also due to less cumulation due to shorter duration of exposure of 2 weeks, as showed by the lower peak brain concentration (Cp) at 48 hours in the parent generation mice. The effect of such short duration of exposure on PCB 180 transfer by other dosing routes across one generation has also been discussed earlier by other studies in rats and humans.\u003csup\u003e26,27\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn summary, the study results prove the existence of hazardous plausibility of neurotoxicological risk of PCB exposures across at least two generations after inhalation exposures. These risks could get amplified in human workers both via indoor (occupational) and environmental exposures and be passed on to next generations translating onto behavioral, neurological and cognitive imbalances and disorders. Such deficits have been assessed in human participants indoor and after environmental exposures also. \u003csup\u003e28,29,30,31\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe probable route of trans-generational transfer of PCBs would be lactational one as compared to the placental route. These changes in anthropometric outcomes (Supplementary Table\u0026nbsp;3) correlate well with alterations in brain Noradrenaline (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and behavior scores in social activity (Supplementary Table\u0026nbsp;4, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) over 14 day exposures, with an additional 7-day observation period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis pilot study provides proof-of-concept evidence on the causality of neurobehavioural deficits in mice as caused by occupational exposures of parent mice via inhalational routes at a dose of 4 mg/cu m. Moreover, this could be due to transfer of PCB congeners across a generation by lactational concentrations due to their basic nature and higher lipophilicities. Although the P generation sample size was lower than the recommended size for statistical significance, the study outcomes are biologically significant and as per the standard CPCSEA guidelines for animal housing, which were also applied to housing inside the inhalational chamber, the sample size of parents were kept minimal for ensuring animal welfare. This pivotal study yields valid and robust results of the epigenetic transgenerational theory of endocrine disruptors as etiological factors in disease pathophysiology in rodents, for confirmation in further studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAhR = Aryl Hydrocarbon receptor; AVMA = American Veterinary Medical Association; ANOVA = Analysis of Variance; BAL = Bronchoalveolar Lavage; BMD = Bench Mark Dose; CPCSEA = Committee for the Purpose of Control and Supervision of Experiments on Animals; EPA = Environment Protection Agency; GD = Gestational Day; H\u0026amp;E = Haematoxylin \u0026amp; Eosin; HPLC = High Performance Liquid Chromatography; IAEC = Institutional Animal Ethics Committee; LLOQ = Lower Limit of Quantification; OECD = Organization for Economic Cooperation and Development; PCB = Polychlorinated Biphenyl; PEL = Permissible Exposure Limit; PND = Postnatal Day; UPLC-DAD = Ultra-High Performance Liquid Chromatography-Diode Array Detector\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe author would also like to acknowledge the facilities given by PSG Institute of Medical Sciences \u0026amp; Research, Coimbatore for the success of the work. The author would like to acknowledge Dr. Ramalingam Sankaran (**deceased), former Dean and Professor of Pharmacology, for his untiring efforts in provision of essential facilities and administrative guidance in the PSG Center for Molecular Medicine \u0026amp; Therapeutics, useful in the execution of the work undertaken in the laboratory. The author acknowledges his role as the Guide for PhD work, of which this work is a key component. The authors like to acknowledge Dr S. Vidhiyalakshmi, Faculty in the department of Pathology, for her help and assistance rendered in staining of lung sections after BAL study sampling.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors state that the study was self-funded by the authors, and declare that no other sources of extramural funding exist.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics \u0026amp; Regulatory Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were followed in this animal study after prior IAEC approval in compliance with the CPCSEA guidelines (2005), AVMA guidelines (2013) and OECD guidance document GD 39.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests during the conception and inception of the paper, including any non-financial and monetary ones.\u003c/p\u003e\n\u003cp\u003eSupplementary material\u003c/p\u003e\n\u003cp\u003eSupplementary material (Supplementary Tables 1-4) is available at \u003cem\u003eBrain\u003c/em\u003e online\u0026rsquo;.\u0026nbsp;\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgency for Toxic Substances and Disease Registry, Toxicological profile for polychlorinated biphenyls (PCBs), November 2000, accessed at https://www.atsdr.cdc.gov/ToxProfiles/tp17.pdf.\u003c/li\u003e\n\u003cli\u003eErin N. Jackson,Sean E. Thatcher,NikaLarian,VictoriaEnglish,SonySoman,Andrew J. Morris,JiayingWeng,ArnoldStromberg,Hollie I. Swanson,KevinPearson,and Lisa A. Cassis, 2019, Effects of Aryl Hydrocarbon Receptor Deficiency on PCB-77-Induced Impairment of Glucose Homeostasis during Weight Loss in Male and Female Obese Mice; Environmental Health Perspectives 127:7 CID: 077004 https://doi.org/10.1289/EHP4133.\u003c/li\u003e\n\u003cli\u003eSven Burreau, YngveZeb\u0026uuml;hr, Dag Broman, RashaIshaq, Biomagnification of PBDEs and PCBs in food webs from the Baltic Sea and the northern Atlantic Ocean, Science ofTheTotalEnvironment,Volume366,Issues2\u0026ndash;3,2006,Pages659-672,ISSN 0048-9697, https://doi.org/10.1016/j.scitotenv.2006.02.005.\u003c/li\u003e\n\u003cli\u003eBell MR. Endocrine-disrupting actions of PCBs on brain development and social and reproductive behaviors. CurrOpinPharmacol. 2014 Dec;19:134-44. doi: 10.1016/j.coph.2014.09.020. Epub 2014 Oct 10. PubMed PMID: 25310366; PubMed Central PMCID: PMC4298313.\u003c/li\u003e\n\u003cli\u003eMaria C. Ferrante, Paola Amero, Anna Santoro, Anna Monnolo, Raffaele Simeoli, Francesca Di Guida, Giuseppina MattaceRaso, Rosaria Meli, Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes, Toxicology and Applied Pharmacology, Volume 279, Issue 3, 2014, Pages 401-408, ISSN 0041-008X, https://doi.org/10.1016/j.taap.2014.06.016.\u003c/li\u003e\n\u003cli\u003eCurran, Christine \u0026amp; W Nebert, Daniel \u0026amp;Genter, Mary \u0026amp; V Patel, Krishna \u0026amp;Schaefer, Tori \u0026amp; Skelton, Matthew \u0026amp; T Williams, Michael \u0026amp; Vorhees, Charles. (2011).In Utero and Lactational Exposure to PCBs in Mice: Adult Offspring Show Altered Learning and Memory Depending on Cyp1a2 and Ahr Genotypes. Environmental health perspectives. 119. 1286-93. 10.1289/ehp.1002965.\u003c/li\u003e\n\u003cli\u003ePeter Thorne, Matt Ampleman, Xin Hu, Andrea Adamcakova-Dodd, Keri Hornbuckle; Uptake of inhaled polychlorinated biphenyls (PCBs) in a human longitudinal cohort study and animal inhalation studies; European Respiratory Journal Sep 2015, 46 (suppl 59) PA4096; DOI: 10.1183/13993003.congress-2015.PA4096.\u003c/li\u003e\n\u003cli\u003eDakeishi M, Murata K, Tamura A, Iwata T., Relation between benchmark dose and no-observed-adverse-effect level in clinical research: effects of daily alcohol intakeon blood pressure in Japanese salesmen, Risk Anal. 2006 Feb;26(1):115-23.\u003c/li\u003e\n\u003cli\u003eLynne T. Haber, Michael L. Dourson, Bruce C. Allen, Richard C. Hertzberg, Ann Parker, Melissa J. Vincent, Andrew Maier \u0026amp; Alan R. Boobis (2018) Benchmark dose (BMD) modeling: current practice, issues, and challenges, Critical Reviews in Toxicology, 48:5, 387-415, DOI: 10.1080/10408444.2018.1430121.\u003c/li\u003e\n\u003cli\u003eJ. Allen Davis, Jeffrey S. Gift, Q. Jay Zhao, Introduction to benchmark dose methods and U.S. EPA\u0026apos;s benchmark dose software (BMDS) version 2.1.1, Toxicology and Applied Pharmacology, Volume 254, Issue 2, 2011, Pages 181-191, ISSN 0041- 008X, https://doi.org/10.1016/j.taap.2010.10.016.\u003c/li\u003e\n\u003cli\u003eFilipsson AF, Sand S, Nilsson J, Victorin K. The benchmark dose method--review of availablemodels,andrecommendations forapplication inhealthriskassessment. Crit Rev Toxicol. 2003;33(5):505-42. Review. PubMed PMID: 14594105.\u003c/li\u003e\n\u003cli\u003eCPCSEA, Indian Journal of Pharmacology 2003;35:257-274, accessed at http://www.ijp-online.com .\u003c/li\u003e\n\u003cli\u003eOECD, Guidance Document 39: Guidance on inhalational toxicity studies, 2/e, July 2018, accessed at http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2009)28/rev1\u0026amp;doclanguage=en.\u003c/li\u003e\n\u003cli\u003eAVMA, AVMA Guidelinesfor the Euthanasiaof Animals: 2013 Edition, ISBN978-1-882691 210, Version 2013.0.1, accessed at https://www.avma.org/KB/Policies/Documents/euthanasia.pdf.\u003c/li\u003e\n\u003cli\u003eRamanujam, N, Sivaselvakumar, M, Ramalingam, S. Fast and parallel determination of PCB 77 and PCB 180 in plasma using ultra performance liquid chromatography with diode array detection: A pharmacokinetic study in Swiss albino mouse. Biomedical Chromatography. 2017; 31:e4000. https://doi.org/10.1002/bmc.4000.\u003c/li\u003e\n\u003cli\u003eNarayanan, R., Muthusamy, S., Lakshmi, S.V. \u003cem\u003eet al. \u003c/em\u003eDesign and development of whole-body rodent inhalation chamber for exposure to Aroclor 1232 in Swiss albino mice. \u003cem\u003eInt. J. Environ. Sci. Technol. \u003c/em\u003e(2021). https://doi.org/10.1007/s13762-021- 03515-8.\u003c/li\u003e\n\u003cli\u003eOECD(2009),TestNo.436:AcuteInhalation Toxicity\u0026ndash; Acute ToxicClassMethod, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264076037-en..\u003c/li\u003e\n\u003cli\u003eGuo YL, Lambert GH, Hsu CC. Growth abnormalities in the population exposed in utero and early postnatally to polychlorinated biphenyls and dibenzofurans. Environ Health Perspect. 1995; 103 (SUPPL. 6): 117\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eCurran CP, Nebert DW, Genter MB, Patel K V., Schaefer TL, Skelton MR, et al. In uteroandlactationalexposuretoPCBs inmice:Adultoffspringshow alteredlearning and memory depending on CYP1A2 and AHR genotypes. Environ Health Perspect. 2011; 119(9): 1286\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003eSafe S. Toxicology, structure-function relationship, and human and environmental health impacts of polychlorinated biphenyls: Progress and problems. Environ Health Perspect. 1993; 100: 259\u0026ndash;68.\u003c/li\u003e\n\u003cli\u003eChevrier J, Eskenazi B, Holland N, Bradman A, Barr DB. Effects of exposure to polychlorinated biphenyls and organochlorine pesticides on thyroid function during pregnancy. Am J Epidemiol. 2008; 168(3): 298\u0026ndash;310.\u003c/li\u003e\n\u003cli\u003eCarpenter DO, Hussain RJ, Berger DF, Lombardo JP, Park HY. Electrophysiologic and behavioral effects of perinatal and acute exposure of rats to lead and polychlorinated biphenyls. Environ Health Perspect. 2002; 110 (SUPPL. 3): 377\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eCromwell HC, Johnson A, Mcknight L, Horinek M, Burt S, Jolous-jamshidi B, et al. Conditioning in Rat Pups. 2014; 91(5): 658\u0026ndash;66.\u003c/li\u003e\n\u003cli\u003eMiyazaki W, Iwasaki T, Takeshita A, Tohyama C, Koibuchi N. Identification of the functional domain of thyroid hormone receptor responsible for polychlorinated biphenyl-mediated suppression of its action in vitro. Environ Health Perspect. 2008; 116(9): 1231\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eQuaak I, Brouns MR, van de Bor M. The dynamics of Autism Spectrum Disorders: How neurotoxic compounds and neurotransmitters interact. Int J Environ Res Public Health. 2013; 10(8): 3384\u0026ndash;408.\u003c/li\u003e\n\u003cli\u003eEspenBorg\u0026aring; Johansen, Monica Knoff, FrodeFonnum, Per LeinesLausund, S Ivar Walaas, Grete W\u0026oslash;ien, TerjeSagvolden, Postnatal exposure to PCB 153 and PCB 180, but not to PCB 52, produces changes in activity level and stimulus control in outbred maleWistar Kyoto rats, Behavioral and Brain Functions, 2011, Volume 7, Number 1, Page 1.\u003c/li\u003e\n\u003cli\u003eYakushiji, T\u0026amp;Watanabe, I \u0026amp;Kuwabara, K \u0026amp;Tanaka, R \u0026amp;Kashimoto, T\u0026amp;Kunita, N \u0026amp; Hara, I.. Rate of decrease and life of polychlorinated biphenyls (PCB) in the blood of mothers and their children exposed to PCB. Archives of environmental contamination and toxicology. 1984. 13. 341-5. 10.1007/BF01055285.\u003c/li\u003e\n\u003cli\u003eLee DH, Jacobs DR, Porta M. Association of serum concentrations of persistent organic pollutants with the prevalence of learning disability and attention deficit disorder. Journal of Epidemiology \u0026amp; Community Health. 2007 Jul 1;61(7):591-6.\u003c/li\u003e\n\u003cli\u003eChen YC, Guo YL, Hsu CC, Rogan WJ. Cognitive development of Yu-Cheng (\u0026apos;Oil Disease\u0026apos;) children prenatally exposed to heat-degraded PCBs. Jama. 1992 Dec 9;268(22):3213-8.\u003c/li\u003e\n\u003cli\u003eHolmes AK, Rubin C, Marcus M, Kieszak S, Jones RW, Golding J, McGeehin M. A pilot study of chemical exposures among mothers and children enrolled in the Avon Longitudinal Study of Parents and Children. InAnnual Meeting.\u003c/li\u003e\n\u003cli\u003eMarek RF, Thorne PS, Wang K, DeWall J, Hornbuckle KC. PCBs and OH-PCBs in serum from children and mothers in urban and rural US communities. Environmental science \u0026amp; technology. 2013 Apr 2;47(7):3353-61.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figure 1","content":"\u003cp\u003eSupplementary Figure 1 is not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inhalational toxicity, Polychlorinated Biphenyl (PCB), trans-generational toxicity, occupational hazard assessment, developmental toxicity","lastPublishedDoi":"10.21203/rs.3.rs-4820257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4820257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAroclor 1232 is a commercial mixture of polychlorinated biphenyls with occupational toxicological implications as it contains semi-volatile congeners like PCB 77, along with highly lipophillic ones like PCB 180. We aimed to assess the trans-generational behavioral toxicities of this complex mixture in mice by inhalational dosing chamber. We also aimed to evaluate the penetrability of PCBs across placental and lactational routes in mice in this study.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe assessed the probable route of penetration in the first generation of litters using behavioral scores as surrogate markers of developmental toxicity. We also quantified plasma concentrations of key PCBs and anthropometric parameters for trans-generational comparability.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePCBs are responsible for behavioral toxicities across one generation of mice by occupational exposures via the inhalational route. Behavioral scores of F1 and F2generation mice indicated as surrogate endpoints that PCBs are concentrated in lactating milk more than placental route in the next generation of mice. No significant lethality or effects on anthropometrical parameters were detected across one generation although the congeners were detected in plasma of the litters when they were 12\u0026ndash;14 weeks age.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePCBs may pose both indoor and outdoor occupational hazard by inhalational routes and cause behavioral deficits across one generation by transfer via lactational routes. PCB 77 is more evident of penetrating trans-generationally and produces behavioral toxicities in subsequent generations. They definitely carry inhalational hazard in workplaces and outdoor as environmental pollutants and neuroendocrine disruptors.\u003c/p\u003e","manuscriptTitle":"Transgenerational toxicity of Aroclor 1232 by inhalational route in mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-10 21:01:41","doi":"10.21203/rs.3.rs-4820257/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c516fed-0194-4216-b01e-21b24169da3b","owner":[],"postedDate":"September 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-14T10:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-10 21:01:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4820257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4820257","identity":"rs-4820257","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}