Fat depot-specific accumulation of benzo(a)pyrene metabolites in rats fed with western diet and its implications for colon cancer.

Aramandla Ramesh: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization. Olugbemiga Ogunkua: Writing – review & editing, Methodology. Markis' D. Hamilton: Writing – review & editing, Software, Data curation. Samuel E. Adunyah: Writing – review & editing, Supervision, Funding acquisition. Anthony E. Archibong: Writing – review & editing, Methodology. Kenneth J. Harris: Writing – review & editing, Methodology, Formal analysis. Kelly L. Harris: Writing – review & editing, Writing – original draft, Methodology, Funding acquisition, Formal analysis.

This research has received financial support from the 10.13039/100000002 National Institutes of Health grants 1F31ESO2407901 , 1UG3HG013248–01 , 5U54CA163069–04 , R25GM160671 , GM151274 , and G12RR03022 , 10.13039/100000015 Department of Energy grants DE EM0005266 , DOE-BSRA G-SOW-A−02557 , DOE-BSRA G-SOW-A−02689 , the 10.13039/100000001 National Science Foundation grants 2229521 OSA00000158 , DBI−2412389 , and the 10.13039/100000048 American Cancer Society grant DICRIDG−21–071–01 .

The weights of brown and white adipose tissue masses retrieved from the various anatomical locations were examined to identify if diet type and B(a)P dose-related changes had an influence on adipose tissue weights in PIRC rats. As regards white adipose tissue (WAT), the inguinal, retroperitoneal, epididymal, subcutaneous fats, mesenteric and visceral adipose masses showed statistically significant differences ( p  < 0.001) in tissue weights among the three B(a)P dose groups within the RD and WD -fed groups ( Fig. 2  A & 2 B). Among the different subtypes of WAT, the inguinal, mesenteric and visceral fats registered higher weights at 50- and 100 µg B(a)P/kg bw groups (2 A). Between the two diet categories, the adipose tissue masses of rats that received WD were higher ( p  < 0.001) compared to their counterparts that received RD ( Fig. 2  A & 2 B). Fig 2 A. Mean white adiose tissue weights of PIRC rats from Regular Diet (RD; 5% fat content) and Western Diet (WD; 58% fat content) groups (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Weight of fat depots from PIRC rats following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD (AIN−76A diet) or WD. Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. 2B. Mean brown adipose tissue weights of PIRC rats from Regular Diet (RD; 5% fat content) and Western Diet (WD; 58% fat content) groups (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Weight of fat depots from PIRC rats following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD. Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. A. Mean white adiose tissue weights of PIRC rats from Regular Diet (RD; 5% fat content) and Western Diet (WD; 58% fat content) groups (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Weight of fat depots from PIRC rats following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD (AIN−76A diet) or WD. Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. 2B. Mean brown adipose tissue weights of PIRC rats from Regular Diet (RD; 5% fat content) and Western Diet (WD; 58% fat content) groups (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Weight of fat depots from PIRC rats following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD. Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. The brown adipose tissue (BAT) showed a different trend. The mediastinal, cervical, interscapular, and perirenal fats showed no significant differences in tissue weights among the three B(a)P dose groups within the RD- and WD-fed groups except for perirenal fat, where there was a statistically significant difference in tissue mass between 25- and 100 µg B(a)P/kg bw ( Fig. 2 B). The perirenal fat weighed more than the mediastinal and cervical fats ( p  < 0.001), but slightly higher than the interscapular fat, which was statistically insignificant. The percent lipid in WAT and BAT is shown in Table 1 . The % lipid in WAT is greater ( p  < 0.001) than BAT. Among the WAT, lipid % was high in visceral and mesenteric fat ( p  < 0.001), while in BAT, the perirenal fat registered greater lipid % ( p  < 0.001). Table 1 Extractable lipid content in different adipose depots of PIRC rat. Adipose tissue Lipid content (% Mean + SE) RD + B(a)P WD + B(a)P RD only 25 µg B(a)P 50 µg B(a)P 100 µg B(a)P WD only 25 µg B(a)P 50 µg B(a)P 100 µg B(a)P /kg bw /kg bw /kg bw /kg bw /kg bw /kg bw WAT Visceral fat 70 + 1.0 72 + 0.8 75 + 1.2 76 + 1.5 79 + 1.3 81 + 1.7 85 + 1.5 88 + 1.2 Mesenteric fat 69 + 0.7 70 + 1.0 73 + 1.4 75 + 0.8 78 + 0.5 79 + 1.0 83 + 0.8 86 + 1.0 Subcutaneous fat 55 + 0.9 57 + 1.1 58 + 0.6 61 + 1.1 61 + 1.2 62 + 1.4 65 + 1.2 70 + 0.8 Epididymal fat 65 + 1.2 66 + 0.4 69 + 0.8 72 + 1.7 75 + 0.6 78 + 1.5 80 + 1.8 83 + 1.5 Retroperitoneal fat 63 + 0.6 64 + 1.3 66 + 1.0 70 + 1.1 71 + 0.9 75 + 1.0 77 + 1.2 80 + 1.2 Inguinal fat 59 + 0.8 58 + 1.0 59 + 0.6 61 + 1.2 63 + 0.7 65 + 0.8 68 + 0.9 75 + 0.7 BAT Mediastinal fat 32 + 1.6 31 + 1.1 30 + 1.0 31 + 0.9 40 + 0.6 41 + 0.5 41 + 0.8 45 + 1.7 Cervical fat 28 + 0.7 31 + 0.4 33 + 0.8 33 + 1.1 35 + 0.8 37 + 0.8 40 + 1.1 43 + 1.5 Interscapular fat 27 + 0.9 30 + 0.4 32 + 0.7 33 + 1.4 37 + 0.4 41 + 0.5 43 + 0.75 47 + 0.8 Perirenal fat 36 + 1.6 38 + 1.3 40 + 1.1 44 + 0.5 51 + 0.8 53 + 1.5 55 + 1.5 57 + 0.4 Fat content of adipose tissues was determined gravimetrically following the method of Association of Analytical Chemists [29] . SE: Standard error; RD: Regular diet; WD: Western diet; WAT: White adipose tissue; BAT: Brown adipose tissue. Extractable lipid content in different adipose depots of PIRC rat. Fat content of adipose tissues was determined gravimetrically following the method of Association of Analytical Chemists [29] . SE: Standard error; RD: Regular diet; WD: Western diet; WAT: White adipose tissue; BAT: Brown adipose tissue. The number of colon polyps was higher in B(a)P-treated PIRC rats, which received the WD in comparison to their counterparts, which were on RD ( Fig. 2 B; p  < 0.001). A dose response relationship was seen in rats as regards colon polyp numbers with the highest number of polyps recorded in rats receiving WD and treated with 100 µg B(a)P/kg bw ( Fig. 3 ; ( p  < 0.001). Fig 3 Polyp counts in the colon of PIRC rat following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. RD: regular diet; WD: Western diet. Polyp counts in the colon of PIRC rat following treatment with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Rats that were fed with RD or WD but received no B(a)P served as controls. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. RD: regular diet; WD: Western diet. Rats that ingested B(a)P + WD registered greater levels of B(a)P metabolites both in their plasma and WAT compared to those that received B(a)P + RD ( Fig. 4  A & 4 B). Rats registered greater concentrations of B(a)P metabolites in adipose tissue compared to plasma at all doses tested in each one of the diet types. The B(a)P metabolite concentrations in WAT were also found to be B(a)P dose - dependent. A significant increase ( p  < 0.001) in metabolite concentrations in plasma and WAT of rats that received 50- and 100 µg B(a)P/kg bw compared to 25 µg B(a)P/kg bw was observed ( Fig. 4  A). The B(a)P metabolite concentrations in BAT of rats that were fed with RD showed a similar pattern to that observed in WAT albeit the metabolite concentrations were low ( Fig. 4 B). Rats that were on WD showed greater ( p  < 0.001) concentrations of B(a)P metabolites when compared to their counterparts that received the same dosage of B(a)P but provided with the RD. As regards the relationship between concentrations of B(a)P metabolites in plasma and fat, a strong correlation was found between plasma and visceral fat (r = 0.80), plasma and mesenteric fat (r = 0.74) in rats that were fed WD in the 50- and 100 µg B(a)P/kg bw in our studies. Fig 4 A. The distribution of B(a)P total metabolite concentrations in plasma, and WAT as a function of administered B(a)P dose, diet type, and fat categories. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. WAT: White Adipose Tissue; RD: Regular Diet; WD: Western Diet. 4B. The distribution of B(a)P total metabolite concentrations in plasma, and BAT as a function of administered B(a)P dose, diet type, and fat categories. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. BAT: Brown Adipose Tissue; RD: Regular Diet; WD: Western Diet. A. The distribution of B(a)P total metabolite concentrations in plasma, and WAT as a function of administered B(a)P dose, diet type, and fat categories. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. WAT: White Adipose Tissue; RD: Regular Diet; WD: Western Diet. 4B. The distribution of B(a)P total metabolite concentrations in plasma, and BAT as a function of administered B(a)P dose, diet type, and fat categories. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. The black solid lines and symbols indicate statistical significance among controls and B(a)P treatment groups in each diet type. The blue dotted lines and symbols indicate statistical significance among controls and B(a)P treatment groups in WD compared to RD. Annotations denote statistical significance (* p  < 0.0001; # p  < 0.005) among diets and B(a)P doses used. BAT: Brown Adipose Tissue; RD: Regular Diet; WD: Western Diet. When the B(a)P metabolite concentrations in WAT were grouped according to the fat sub-type, the following accumulation pattern emerged. Visceral > Mesenteric > subcutaneous > epididymal > retroperitoneal > inguinal across all dose groups and diet types ( p   interscapular fat > mediastinal fat > cervical fat ( Fig. 4 B). However, the differences in B(a)P metabolite concentrations among the BAT sub-types were not statistically significant. The B(a)P metabolite profiles and proportions are shown in Figs. 5  A & 5 B. The B(a)P metabolites identified include B(a)P 4,5-diol; B(a)P 7,8-diol; B(a)P 9,10-diol; B(a)P 3,6-dione; B(a)P 6,12-dione; 3(OH) B(a)P and 9(OH) B(a)P. The diol and dione metabolites are reactive metabolites, and the hydroxy metabolites are detoxification metabolites. The metabolite composition in visceral fat is depicted in Fig. 5  A and this adipose depot registered greater concentrations of metabolites compared to other white fat categories. The metabolite composition in perirenal fat as an example of BAT is shown in Fig. 5 B. In BAT, the 3(OH) and 9(OH) B(a)P registered greater proportions compared to the diols and dione metabolites. Fig 5 A. The distribution of B(a)P metabolite types in plasma, and visceral fat (a representative WAT) as a function of B(a)P administered dose and diet type. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. WAT: White Adipose Tissue; RD: Regular Diet; WD: Western Diet. 5B. The distribution of B(a)P metabolite types in plasma, and perirenal fat (a representative BAT) as a function of B(a)P administered dose and diet type. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. BAT: Brown Adipose Tissue; RD: Regular Diet; WD: Western Diet. A. The distribution of B(a)P metabolite types in plasma, and visceral fat (a representative WAT) as a function of B(a)P administered dose and diet type. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. WAT: White Adipose Tissue; RD: Regular Diet; WD: Western Diet. 5B. The distribution of B(a)P metabolite types in plasma, and perirenal fat (a representative BAT) as a function of B(a)P administered dose and diet type. Samples were collected from PIRC rats that were treated with 25-, 50-, and 100 µg B(a)P/kg body weight for 60 days via oral gavage and consumption of RD or WD (n = 10 each for controls, diet types, and corresponding B[ a ]P dose groups). Samples were processed using liquid-liquid extraction and metabolite concentrations were determined by HPLC and validated using standards obtained from the National Cancer Institute. BAT: Brown Adipose Tissue; RD: Regular Diet; WD: Western Diet.

Seven-week-old male PIRC rats (purchased from Taconic [Hudson, NY] or bred in house at the University of Missouri) which are genetically altered to mimic sporadic cancer as seen in humans, were used for the studies reported below. Only male rats were chosen to ascertain whether epididymal fat being a specialized WAT act as an energy depot and an active endocrine and metabolic organ could influence colon cancer progression in males. Animals were cared for and housed in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal care facility according to the recommendations established in the NIH Guide for the Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines [26] . The PIRC rats were maintained on a 12/12-hour light/dark cycle (lights on at 0600 h) and allowed free access to either the AIN−76A diet (regular diet; RD; 5% fat content) or WD (58% fat content) and water. The diets were purchased from Research Diets, Inc. (New Brunswick, NJ). The PIRC rats were housed in groups of 2 per cage and allowed a seven-day acclimation period before they were randomly assigned to the following treatment categories: I-AIN−76 diet (RD) only (n = 10), II- WD only (n = 10), III-B(a)P (25 µg/kg bw) + RD (n = 10), IV-B(a)P (50 µg/kg bw) + RD (n = 10), V-B(a)P (100 µg/kg bw) + RD (n = 10), VI-B(a)P (25 µg/kg bw) + WD (n = 10), VII-B(a)P (50 µg/kg bw) + WD (n = 10), VIII- B(a)P (100 µg/kg) + WD (n = 10). The number of rats used were determined after conducting a “statistical power analyses” using PS software (Dupont and Plummer 1990). Rats were administered B(a)P (97% pure, Sigma Chemical Co., St. Louis, MO), dissolved in research grade tricaprylin (Sigma), daily via oral gavage at a volume of 300 µL for 60 days. The overall wellbeing of rats in both control and treatment groups was checked twice a day (including holidays and weekends). Rat body weights and food consumption were monitored periodically. Benzo(a)pyrene being a potential carcinogen was handled in accordance with NIH guidelines [27.] At the end of exposure period, rats were anaesthetized using isoflurane (3%). Blood samples were collected by cardiac puncture using Vacutainer double-ended needles inserted directly into Vacutainer blood collection tubes containing sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Post blood collection, rats were euthanized using excess isoflurane (6%), adipose and other tissues of interest were retrieved. Rats that were fed with the above-mentioned diets but received no B(a)P (categories I & II) served as controls. Brown adipose tissues were dissected from four regions namely: mediastinal, cervical, interscapular, and perirenal regions. Mediastinal adipose tissue lies among mediastinal blood vessels, heart, trachea and esophagus. Cervical adipose tissue lies around the neck. Interscapular adipose tissue lies in the area between the shoulder blades. Perirenal adipose tissue constitutes the fat surrounding the kidneys. White adipose tissues dissected from six regions include: visceral, mesenteric, subcutaneous, inguinal, retroperitoneal, and epididymal adipose tissues. Visceral adipose tissue is located inside the abdominal cavity surrounding internal organs such as stomach, intestines, pancreas, liver etc. Mesenteric adipose tissue is located along the mesentery, traversing from stomach to colon. Subcutaneous adipose tissue is located beneath the skin as a continuous layer with varying thickness. Inguinal adipose tissue refers to the subcutaneous fat tissue between the lower part of the rib cage and the thighs. Retroperitoneal adipose tissue lies along the posterior wall, extending from the kidney to the hip region. Epididymal adipose tissue constitutes the fat tissue surrounding the epididymis. The location of various fat depots is shown in Fig. 1 . Fig 1 The distribution of widely studied brown- and white adipose depots in the PIRC rat. This illustration enables readers to grasp the location of these two important fat categories. BAT: Brown Adipose Tissue; WAT: White Adipose Tissue. The distribution of widely studied brown- and white adipose depots in the PIRC rat. This illustration enables readers to grasp the location of these two important fat categories. BAT: Brown Adipose Tissue; WAT: White Adipose Tissue. A volume of 250 μL of plasma or 200–250 mg of tissue were each homogenized in 500 mL of Tris-sucrose-EDTA buffer (0.25 M; pH 7.4). Ten micro liters of sodium dodecyl sulfate (1%) were added to the mixture, vortexed for 1 min and the homogenate was subjected to a liquid-liquid extraction using methanol, deionized water, and chloroform. The extracts were concentrated, filtered and analyzed as mentioned in Ramesh et al. [28] . Fat and plasma sample were analyzed using a High-Performance Liquid Chromatograph, (HPLC; Model 1200, Agilent, Wilmington, DE) equipped with a HP1046 fluorescence detector [28] . The identification and quantification of the metabolites were accomplished by comparing the retention times and peak areas of all samples with that of standards. The standards were purchased from the NCI Chemical Carcinogen Repository within the Midwest Research Institute Repository located in Kansas City, MO. The fat content was determined gravimetrically using the Association of Official Analytical Chemists (AOAC) method [29] . During chloroform-methanol extraction of tissues for B(a)P metabolites followed by organic layer separation, 1 mL of each aliquot was dried at 100°C for 16 h prior to determining the extractable lipid content. Data on adipose tissue weights in rats were analyzed by one-way analysis of variance (ANOVA) to determine the effect of diet type, and B(a)P dose. Similarly, data on B(a)P metabolite concentrations, and colon polyps were analyzed by one-way ANOVA to determine the effect of diet type, B(a)P dose and fat depot type. The differences among means were determined with Bonferroni’s post-hoc test. The criterion for statistical significance was set at p  < 0.05.

It has been reported that for lipophilic chemicals, equilibrium exists between adipose tissue lipids and serum lipids [30] contributing to similar partition coefficients, and there is no diffusion limitation in adipose tissues [31] . Published reports also indicated that blood PAH concentrations are predictive of fat PAH concentrations [13] , [32] . If this assumption is correct, there should not be much difference in B(a)P metabolite levels in adipose tissue and plasma. However, a comparison of the concentrations of B(a)P in adipose tissue and plasma quantified in the present study revealed high concentrations of B(a)P in adipose tissue compared to plasma, indicating a greater degree of partitioning into this tissue. The preferential partitioning of B(a)P into fat tissues and the consequent accumulation could be attributed to its affinity for lipids [33] . Our finding of significant accumulation of B(a)P metabolites in adipose masses suggests that adipose tissue serves as a storage reservoir for this carcinogen. Higher concentrations of priority PAHs were measured in the adipose tissue (visceral, mesenteric, and subcutaneous) compared to serum of Polish and French bariatric patients [13] . Further, high levels of PAHs such as naphthalene and phenanthrene were detected in the adipose tissues of Korean women [23] . Similarly, high concentrations of B(a)P, fluoranthene, phenanthrene, fluorene, and several other PAHs were found in the adipose tissues of people in the Southeast China [14] . Analyses of adipose tissues and body fluids collected from autopsied individuals by our team [34] and others [32] also revealed substantial accumulation of PAHs. The elevated B(a)P metabolite concentrations in WAT than BAT could be attributed to variations in lipid content between these two biological matrices measured in our study. Our studies agree with the lipid levels reported in literature for these two depots. (80–90% fat content in WAT versus 40–50% in BAT; [35] ; 87.4% in WAT; [36] ). Spectral computed tomography studies conducted in Wistar rats also confirmed lower fat concentration in BAT compared to WAT [37] . Circulating B(a)P metabolites showed a significant positive relationship ( p  < 0.0001) with visceral fat mass and mesenteric fat mass in our study. This correlation was more pronounced in rats that received B(a)P through WD at high B(a)P dose groups (50- and 100 µg B(a)P/kg bw) compared to the low dose group (25 µg B(a)P/kg bw). The partitioning of B(a)P from plasma to adipose tissue and its elimination from adipose tissue is linked to its pharmacokinetic characteristics. Using a rat model, Heredia-Ortiz et al. [33] investigated the pharmacokinetic behavior of B(a)P. These authors demonstrated that t1/2 (elimination half-life) of B(a)P parent compound was found to be 27.2 hrs, while one of its metabolites, 3(OH) was 7.1 hrs. The uptake of B(a)P (transfer rate of B(a)P) from the blood compartment to the adipose tissue was found to be slow (t 1/2 3.7 min). Pharmacokinetic modeling of B(a)P in Sprague Dawley rats revealed that the adipose tissue: blood partition coefficient value was found to be high (next only to lung; [38] ). These findings show the rate-limiting elimination of B(a)P and its metabolites stored in adipose tissues. Significantly enough, our studies also found greater concentrations of B(a)P metabolites in adipose tissues. The partitioning of B(a)P into adipose tissue was further confirmed by the findings of Crowell et al. [39] and Deng et al. [40] , who conducted a PBPK modeling of B(a)P in tissues of rodents and humans respectively. The variations in B(a)P metabolite burden in BAT and WAT reflect the differential deposition, mobilization and function of these two depots. The smaller distribution volume of BAT, low lipid content, lower partition coefficient, and higher blood flow could have resulted in rapid distribution and elimination [41] , [42] of metabolized B(a)P in BAT compared to WAT. Among the different WAT sub-types, both visceral and mesenteric fats registered greater concentrations followed by subcutaneous and epididymal fats. Other findings in human subjects also showed a propensity for greater deposition of PAHs in visceral fat. The PAH body burden showed a strong association with BMI and visceral adipose tissue in Koran women. Residues of PAHs were also found in the visceral adipose tissue of women suffering from endometriosis in Poland [12] . Sousa et al. [43] reported positive correlations between PAHs and some fatty acids in the adipose tissue (subcutaneous) of breast cancer patients undergoing surgical procedures and control subjects undergoing breast reduction mammoplasty in Portugal. Qin et al. [44] also detected PAH accumulation in subcutaneous fat and visceral fat of Hong Kong residents with concentrations more pronounced in visceral fat. PAHs were also detected in the visceral adipose tissues of Chinese patients undergoing abdominal surgery [14] . Data from the NHANES survey conducted between 2001 and 2016 revealed a strong correlation between visceral fat and PAHs and their metabolites such as naphthalene, phenanthrene, 2 naphthalene, 1-phenanthrene, 2-phenanthrene, and 9-fluorene in urine [45] . PAHS were also measured in the omental adipose tissues of gastric cancer patients in France [46] . A strong correlation was found between 1-hydroxypyrene metabolite, and visceral fat in urban dwellers of Iran [47] . The PAH residues in subcutaneous and visceral fat tissues showed a positive correlation with total cholesterol levels [48] . Additionally, surgical leftovers of subcutaneous adipose tissue collected from patients undergoing abdominoplasty, liposuction, and mastopexy procedures in Italy revealed accumulation of pyrene in this tissue [9] . Similarly, PAHs were also detected in the breast, arm (subcutaneous), and abdominal (visceral) adipose tissues harvested from patients undergoing abdominoplasty, liposuction, and prosthesis removal procedures in Italy [49] . The body burden of PAHs was found to be lower in the serum of obese patients who underwent bariatric surgery [50] . On the contrary, high levels of PAHs were found in subcutaneous compared to visceral adipose tissues of patients undergoing bariatric surgery in Portugal [48] . These findings reiterate the storage capability of mesenteric/visceral adipose tissue, which after rapid weight loss could release sequestered PAHs into the blood stream and contribute to elevated levels of circulating PAHs. Typically, toxicant concentrations are expressed on a wet weight basis i.e. per unit volume or weight of plasma or tissues analyzed. As some toxicants tend to accumulate in lipid-rich tissues, to overcome the difficulties associated with their quantification, lipid normalization (toxicant concentration is divided by plasma or tissue lipid content) is advocated for reporting the concentrations of toxicants [51] , [52] . This approach rests on the assumption that the toxicant concentrations are evenly distributed in all lipid-rich matrices, and the toxicant/metabolite concentration varies in direct proportion to the lipid content of the matrix. Adherence to this isometric relationship assumption between toxicant concentration and lipid concentration increases the likelihood of arriving at erroneous conclusions because not all lipophilic toxicants accumulate in fat in a uniform manner [53] . The differential partitioning of B(a)P in some lipid types [54] , and the various types of lipid extraction methods employed by researchers [55] are additional shortcomings. Therefore, to account for variations in lipid content as a function of BaP dose and adipose tissue type, our data are presented on wet weight basis, but lipid content was also provided ( Table 1 ) for the benefit of readers. The accumulation of PAHs in the adipose tissue results from dietary intake. Co-ingestion of B(a)P and dietary lipids could increase the solubility and absorption of B(a)P [56] . Our prior studies in rodent models have shown that B(a)P in a lipid milieu is effectively delivered to target organs including colon where it interacts with cellular macromolecules that cause damage to DNA and contributed to tumors formation. Apc Min mice that were administered B(a)P in saturated fat registered high polyp numbers in colon, compared to their counterparts that received B(a)P either through unsaturated fat or tricaprylin [57] . PIRC rats that were fed Western diet and received B(a)P through oral gavage also developed high polyp numbers in colon compared to their counterparts that were on regular diet and orally dosed with B(a)P [21] . Geyer et al. [58] suggested that adipose tissue may aid in detoxification by preventing the toxicant parent compound and/or its metabolites reaching the sites of action. Since both WAT and BAT are equipped with drug metabolizing enzymes, they appear to be involved in the metabolism of B(a)P. Galvan et al. [24] reported expression of CYP1B1 in WAT and CYP1A1 in BAT. Conversely, increased expression of CYP1A1 mRNA was found in epididymal, and mesenteric WAT of rats compared to subcutaneous WAT [59] . The B(a)P-treated mice showed a significant increase in CYP1A1 protein and mRNA expression, which may have contributed to formation of B(a)P metabolites (Lou W et al., 2022). Our earlier studies demonstrated an increase in CYP1A1 and CYP1B1 protein and mRNA expression in liver and colon of PIRC rats treated with WD compared to RD [21] . Since visceral (omental) fat covers the gastrointestinal tract thus, concentrations of B(a)P and/or other PAHs in this fat depot are likely to influence the tumor microenvironment. As WAT is proactively involved in lipid metabolism and storage of energy [60] , any change in positive energy balance can lead to expansion of WAT cell size and number to accommodate increased lipid stores through adipose tissue remodeling [61] . The PAH metabolites were also known to inhibit lipolysis increasing the risk of obesity [62] . Using 3T3-L1 and BAT1 cell lines representing white and brown adipocytes respectively, Bright et al. [63] reported that chronic exposure to PAHs promote lipid accumulation and inflammation in adipocytes. Long-term exposure to PAHs-induced oxidative stress and obesity-related chronic inflammation [64] may exacerbate the risk for developing diet-induced cancer [20] , [21] , [57] . One of the mechanisms suggested the involvement of B(a)P and/or other PAHs, even at low doses increase the adipose tissue mass by blocking the adrenergic activation of lipolysis [17] , [65] , [66] . B(a)P has been reported to inhibit PPARγ and inhibit WAT expansion in C57BL/6 mice [67] . It has been reported that long-term intake of high fat diet causes white adipose tissue remodeling and dysfunction [68] , which promotes a chronic inflammatory state, exemplified by elevated expression of proinflammatory markers such as IL1A, IL1B, IL6, IL8, IL36γ and extracellular matrix markers such as MMP9, SPP1, and TNC associated with colon cancer [69] . While our studies have shown a smaller to significant increase in adipose tissue masses of B(a)P-treated PIRC rats, Lou et al. [67] reported a significant decrease in the epididymal and subcutaneous adipose tissue of C57BL/6 mice. This finding indicates that B(a)P exposure inhibits adipose tissue expansion and fat deposition. The discrepancy between Lou et al. [67] and our findings could be due to the difference in B(a)P dose ranges (Lou et al., 0.90 mg/kg body wt/day & 1.80 mg/kg body wt/day versus 25-, 50- and 100 µg/kg in the present study) and exposure duration (Lou et al., 12 weeks versus 9 weeks in our study) employed. Further, the discrepancy in adipose tissue masses between the rat model used in our study (PIRC rat model) and C57BL/6 mice [67] may be explained by the metabolic state of each species of animals used in the studies. Generally, mice have higher metabolism than rats [70] . Hence the reduced epididymal and subcutaneous adipose tissue of c57BL/6 mice compared with the rat model used in this study. The distribution of B(a)P metabolites in visceral fat (a representative of WAT) and perirenal fat (a representative of BAT) reflects the differential biotransformation capabilities of these two adipose depots. In WAT, the diol and dione metabolites were in greater proportion, while in BAT, the 3(OH) and 9(OH) metabolites dominated. Of these metabolites, the detection of B(a)P trans 7,8-dihydrodiol deserves consideration. This metabolite is the precursor of ( + ) anti-B(a)P diol epoxide (BPDE), one of the carcinogenic intermediates of B(a)P [71] . Additionally, we detected B(a)P 3,6- and 6,12-dione metabolites, which were also implicated in cancer development [72] . The levels of diol and dione metabolites registered an increase while the levels of 3(OH) and 9(OH) metabolites registered a decrease in WAT. On the contrary, a reverse trend was noted in the case of BAT. The differential proportion of these metabolites in the total metabolite burden in WAT and BAT suggests impairment of B(a)P detoxification in WAT and enhanced detoxification in BAT. Our findings on increase in B(a)P metabolite levels with WD ingestion in visceral fat are in consonance with our lab’s earlier findings wherein we have shown high concentrations of B(a)P diol and dione metabolites in the colon of Apc Min mice colon cancer model administered with B(a)P in saturated fat [57] and PIRC rat colon cancer model fed with Western diet [20] in a subchronic exposure regimen. These findings further confirm the increased bioactivation of B(a)P in visceral fat, which through various inflammatory pathways could accelerate colon carcinogenesis. Our findings suggest a longer residence time of B(a)P in fat tissues, especially the WAT because of which it requires a longer time to clear from the system. Therefore, it can be expected that long-term exposure to B(a)P leads to its accumulation in WAT contributing to not only obesity and co-morbidities such as colon cancer. Because of the ability of B(a)P to accumulate in adipose tissue, this tissue is considered to afford a protective function as it shelters the parent compounds and/or their metabolites from reaching the target tissue through circulation [73] . This observation raises the question of whether the protective effect rendered by adipose tissue is localized to a specific fat depot, and whether this effect is dose specific. In this regard, physiologically based pharmacokinetic models focusing on B(a)P [39] , [74] , [75] studies are warranted representing various scenarios such as normal feeding, caloric restriction, and providing calorie-rich diet to study the mobilization of B(a)P from fat stores in single exposure and sequential multiple exposure scenarios. Regarding generalizability of findings from this study to females, questions may arise about the selection of male animals as it is well documented that men’s pattern of disease development may not apply to women and for improving health outcomes research, inclusion of both sexes in study design is suggested [76] . While we are cognizant of this recommendation, we opted for male rat models due to the absence of cyclic hormonal variations compared to female rat models in which hormonal cycles influence the B(a)P biotransformation enzymes. The shift in metabolic pathways may alter the metabolic burden of B(a)P in tissues [77] .

During the preparation of this work, the author(s) used AI-assisted technologies to prepare part of Figure 1 (outline of animal body and internal organs) only. AI-assisted technologies were not used in drafting the manuscript.

Obesity has been recognized as a global pandemic, affecting 40% of the population in developed nations. One of the several diseases associated with obesity is colorectal cancer, as demonstrated in human and animal model studies [1] . Colorectal cancer ranks second in terms of its incidence among the various cancers and mortalities associated with this type of cancer [2] . In addition to metabolic syndrome linked to a 13% increased risk for colorectal cancer [3] , consumption of Western diet rich in combustion pollutants such as polycyclic aromatic hydrocarbons (PAHs) has also been implicated in colorectal cancer risk because these compounds can induce inflammation and promote tumor formation [4] . Because 20 or more PAHs were detected in most dietary ingredients [5] , it is conceivable that PAHs will have an impact on obesity due to their lipophilic property and thus sequester in foods that are rich in saturated fats [6] . After dietary intake, PAHs are released from the ingested food components, metabolized, and absorbed in the gastrointestinal tract, undergo hepatic clearance, enter the systemic circulation, and reach tissues in target organs [7] including adipose tissues [8] , [9] . Studies conducted in animal models [10] , livestock [11] , and humans [12] , [13] , [14] demonstrated accumulation of PAHs in the body. One of the prototypical PAH toxicants that has been linked to obesity due to its role in adipogenesis dysregulation and proinflammatory activity is benzo(a)pyrene (B(a)P; [15] ). An epidemiological survey conducted in 1983 in the New York area revealed that higher plasma B(a)P concentrations correlated with higher body mass index in human subjects [16] . Long-term exposure of mice to B(a)P caused a 43% weight gain compared to controls, which could not be reversed upon cessation of B(a)P exposure [17] . Aside from human studies, studies in cell culture and animal model studies also confirm that B(a)P exposure contributes to metabolic diseases such as obesity [18] and liver steatosis [19] . Our previous studies [20] , [21] have established the importance of Polyposis In Rat Colon (PIRC) rat model to study the contribution of Western diet (WD) in conjunction with B(a)P exposure compared to regular diet (RD) to the development of CRC. Our studies have also shown that B(a)P in combination with WD contributes to a greater visceral weight adipose tissue compared to subcutaneous adipose tissue [20] . Since most fatty acids in adipose tissue are derived from diet with less contribution of de novo synthesis (less than 1 g fatty acids per day; [22] ), it is conceivable that diet-borne toxicants like B(a)P sequester in adipose tissue in long-term exposure scenarios. Prior published studies have examined B(a)P in adipose tissue of animal models and humans generally, [6] , [12] , [23] , [24] , but not across systematically defined fat depots in a colon cancer model, which is an important research gap that needs to be addressed. The composition of adipose tissue is not homogenous and varies across anatomical sites in most mammals (reviewed in [25] ) . However, whether the preferential accumulation of B(a)P in various fat depots in relation to plasma have any bearing on tumor development, is not well understood. Therefore, the objective of the present study is to determine the accumulation of B(a)P in the brown and white adipose tissues of the PIRC rat model of colon cancer and its implications in colon carcinogenesis.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.