Frank Inyang

Metabolism, bioavailability, and toxicokinetics of Benzo(α)pyrenein F-344 rats following oral administration

The objective of this study was to evaluate the bioavailability of Benzo(a)pyrene [B(a)p], subsequent to oral exposure. Eight-week-old F-344 rats were dosed orally with 100 mg/kg body weight B(a)p and sacrificed at 0, 0.5, 1.0, 2.0, 4.0. 8.0, 24, 48 and 72 hours post exposure. Blood, liver, reproductive tissues, urine and fecal samples were collected at necropsy and were analyzed for parent B(a)p and metabolites by HPLC with fluorescence detection. Peak levels of B(a)p in plasma occurred 8 hours after exposure (67%) followed by a gradual decrease. Liver retained 10% of the administered B(a)p up to 24 hours following, which the levels dropped during the remaining time periods studied. Twenty-four hours after administration, 45% of the dose was excreted in feces and urine. Metabolite levels in plasma peaked at 24 hours (10%) and decreased to 1% at 72 hours. In the liver, metabolite levels were higher at 8 hours (10%) but were only 3% at 72 hours. Benzo(a)pyrene levels increased after 24 hours in the reproductive organs and constituted 10% of the administered dose at 72 hours. Blood showed high levels of 7,8-diol than 9,10 and 4,5-diols which were high in liver and reproductive organs. Compared to diols, the hydroxy metabolites were detected at high levels in urine and fecal samples. Among the aqueous phase metabolites, glucuronides were at higher levels compared to glutathiones and sulfates. The slow release of unmetabolized B(a)p from reproductive organs and the presence of reactive metabolites in these organs is a matter of concern as they could interfere with gonadal steroid synthesis and release and its regulatory role in gamete production, maturation and function of male animals in a continuous exposure paradigm.

Alteration of pregnancy related hormones and fetal survival in F-344 rats exposed by inhalation to benzo(a)pyrene

The objective of this study was to evaluate the effect of subacute exposure to inhaled benzo(a)pyrene (BaP) on fetal survival and luteal maintenance using timed-pregnant Fisher 344 rats. Prior to assignment of pregnant rats to treatment and control groups, numbers of implantation sites were determined on gestation day (GD) 8 via midventral laparotomy. Subsequently, animals were assigned randomly to three treatment groups and two control groups. Treatment consisted of subacute exposure of rats via inhalation to BaP 25, 75, and 100 micro g/m(3), 4h daily for 10 days (GD-11-20). Control animals were either sham exposed to carbon black (CB) to control for inert BaP carrier or remained unexposed (UNC). Blood samples were collected on days 15 and 17 of gestation via sinus orbital veini-puncture for plasma. Number of pups per litter was determined postpartum and fetal survival rate was expressed as a percentage of the corresponding implantation sites. Radioimmunoassays were used to determine plasma progesterone, estrogen, and prolactin (indirect measurement of decidual luteotropin) concentrations. Fetal survival among BaP-treated rats declined in a dose-dependent manner (25 micro g/m(3), 78.3% per litter; 75 micro g/m(3), 38.0% per litter; 100 micro g/m(3), 33.8% per litter; P<0.05) compared with CB (96.7% per litter) and UNC (98.9% per litter). Plasma progesterone, estrogen, and prolactin concentrations also declined as a result of subacute exposure of rats to BaP compared to controls. These data suggest that inhaled BaP compromised fetal survival and consequently luteotropic activity in the exposed animals.

Disruption of testicular steroidogenesis and epididymal function by inhaled benzo(a)pyrene.

The objective of this study was to evaluate the effect of sub-acute exposure to inhaled benzo(a)pyrene (BaP) on testicular steroidogenesis and epididymal function in Fisher 344 rats. Animals were assigned randomly to two control groups and one experimental group for each exposure regimen. Treatment consisted of sub-acute exposure of rats via inhalation to 25, 75, and 100 microg BaP/m(3), 4 h daily for 10 days. Control animals were either exposed to carbon black (CB; sham) to control for inert BaP carrier or they remained unexposed (UNC). Blood samples were collected immediately after the cessation of exposures (time 0) and at 24, 48, and 72 h post-cessation of exposure, to assess the effect of bioavailable BaP on systemic testosterone and luteinizing hormone (LH) concentrations by radioimmunoassay (RIA). Progressive sperm motility of stored sperm (cauda epididymal sperm) was determined microscopically, while density of stored sperm was determined by hemocytometric counting. Progressive motility of stored sperm was reduced in rats exposed to 75 and 100 microg BaP/m(3) compared with their counterparts that were exposed to 25 microg BaP/m(3) or controls. Plasma testosterone concentrations declined as a result of exposure of rats to 75 microg BaP/m(3) from 0 to 48 h post-termination of exposure compared with controls (P<0.05; treatment x time interaction). This decrease was followed subsequently by a compensatory increase in the plasma concentrations of this steroid at 72 h post-cessation of exposures compared with previous time periods and controls (P<0.05). Increases in the mean plasma LH concentrations were observed in rats exposed to 75 microg BaP/m(3) compared with controls, throughout the time periods studied (P<0.05; treatment x time interaction). These data suggest that sub-acute exposure to inhaled BaP contributes to reduced testosterone concentrations and consequently impaired epididymal function of exposed animals.

MODULATION IN THE DEVELOPMENTAL EXPRESSION PROFILE OF Sp1 SUBSEQUENT TO TRANSPLACENTAL EXPOSURE OF FETAL RATS TO DESORBED BENZO[a]PYRENE FOLLOWING MATERNAL INHALATION

Any alteration of the critical sequence of genes that are required to coordinate the differentiation of cells, the promotion of migration, dendritic arborization, synapse formation, and myelination in the developing nervous system would be expected to have deleterious consequences. The focus of this article is a molecular evaluation of the neurotoxicological effects that result subsequent to the transplacental exposure of fetal rats to desorbed benzo(a)pyrene (BaP) following maternal inhalation. A state-of-the-art, newly designed, fabricated, and tested model aerosol generation system was utilized in these studies. Timed-pregnant Sprague Dawley rats were exposed for 4 h on gestation day 15 of a 21-day gestation period to an acute dose of BaP: carbon black aerosol (100 µg/m3). Controls received carbon black only. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The aerosol exhibited a trimodal distribution with 95% cumulative mass less than 15.85 µm, 90% cumulative mass less than 10 µm, 67.5% cumulative mass less than 2.5 µm and 66.2% cumulative mass less than 1.0 µm. Time-course bioavailability results indicated that greater than 95% of the parent compound is cleared from blood 240 min postexposure. An Sp1 transcription factor consensus sequence was examined by electrophoretic mobility shift analysis of nuclear extracts from various brain regions of resulting pups on postnatal days 3, 5, 7, 10, and 15. It revealed perturbations in the developmental expression profile of Sp1 abundance as a result of nose-only particulate aerosol exposure to the timed-pregnant dam. The data obtained on the temporal and spatial regulation of gene expression in the brain indicate that (1) Sp1 DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a consequence of the transplacental deposition of desorbed BaP to the fetus is in utero neurotoxicity.Any alteration of the critical sequence of genes that are required to coordinate the differentiation of cells, the promotion of migration, dendritic arborization, synapse formation, and myelination in the developing nervous system would be expected to have deleterious consequences. The focus of this article is a molecular evaluation of the neurotoxicological effects that result subsequent to the transplacental exposure of fetal rats to desorbed benzo(a)pyrene (BaP) following maternal inhalation. A state-of-the-art, newly designed, fabricated, and tested model aerosol generation system was utilized in these studies. Timed-pregnant Sprague Dawley rats were exposed for 4 h on gestation day 15 of a 21-day gestation period to an acute dose of BaP:carbon black aerosol (100 microg/m(3)). Controls received carbon black only. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The aerosol exhibited a trimodal distribution with 95% cumulative mass less than 15.85 microm, 90% cumulative mass less than 10 microm, 67. 5% cumulative mass less than 2.5 microm and 66.2% cumulative mass less than 1.0 microm. Time-course bioavailability results indicated that greater than 95% of the parent compound is cleared from blood 240 min postexposure. An Sp1 transcription factor consensus sequence was examined by electrophoretic mobility shift analysis of nuclear extracts from various brain regions of resulting pups on postnatal days 3, 5, 7, 10, and 15. It revealed perturbations in the developmental expression profile of Sp1 abundance as a result of nose-only particulate aerosol exposure to the timed-pregnant dam. The data obtained on the temporal and spatial regulation of gene expression in the brain indicate that (1) Sp1 DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a consequence of the transplacental deposition of desorbed BaP to the fetus is in utero neurotoxicity.

Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene

The objective of this study was to evaluate the reproductive risk associated with exposure of adult male Fisher-344 rats to inhaled benzo(a)pyrene (BaP). Rats were assigned randomly to a treatment or control group. Treatment consisted of sub-chronic exposure of rats via inhalation to 75microgBaP/m(3), 4h daily for 60 days, while control animals were unexposed (UNC). Blood samples were collected immediately after the cessation of exposures (time 0) and subsequently at 24, 48, and 72h, to assess the effect of bioavailable BaP on plasma testosterone and luteinizing hormone (LH) concentrations. Rats were sacrificed after the last blood collection. Testes were harvested, weighed and prepared for histology and morphometric analysis, and cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa. BaP exposure reduced testis weight compared with UNC (mean+/-SE; 2.01+/-0.11 versus 3.04+/-0.16g; P<0.025), and caused significant reductions in the components of the steroidogenic and spermatogenic compartments of the testis. Progressive motility and mean density of stored spermatozoa were reduced (P<0.05). Plasma testosterone concentrations were decreased by two-thirds in BaP-exposed rats throughout the time periods studied compared with those of their UNC counterparts (P<0.05), concomitant with increased concentrations of LH in BaP-exposed rats (P<0.05). These data suggest that sub-chronic exposure to inhaled BaP contribute to reduced testicular and epididymal function in exposed rats.

Toxicokinetics of inhaled benzo[a]pyrene: plasma and lung bioavailability.

Bioavailability and toxicokinetic studies are essential in order to establish dose-response relationships of widely distributed environmental toxicants such as benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon. Fischer 344 rats were exposed for 4 h (via nose-only inhalation) to aerosol exposure concentrations of 0.1, 1.0, and 2.5 mg/m3 of BaP absorbed onto carbon black particles using a state-of-the-art model aerosol generation system. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The average aerosol for the 3 exposure concentrations used in this study exhibited a trimodal distribution with 93% cumulative mass less than 15.85 µm, 89% cumulative mass less than 10 µm, 55.3% cumulative mass less than 2.5 µm, and 38% less than 1 µm. Fifty-five percent of the aerosol had a cumulative mass less than PM2.5Bioavailability and toxicokinetic studies are essential in order to establish dose-response relationships of widely distributed environmental toxicants such as benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon. Fischer 344 rats were exposed for 4 h (via nose-only inhalation) to aerosol exposure concentrations of 0.1, 1.0, and 2.5 mg/m(3) of BaP absorbed onto carbon black particles using a state-of-the-art model aerosol generation system. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The average aerosol for the 3 exposure concentrations used in this study exhibited a trimodal distribution with 93% cumulative mass less than 15.85 microm, 89% cumulative mass less than 10 microm, 55.3% cumulative mass less than 2.5 microm, and 38% less than 1 microm. Fifty-five percent of the aerosol had a cumulative mass less than PM(2.5) and the mass median aerodynamic diameter (MMAD) -/+ geometric standard deviation (GSD) for this mode was 1.7 -/+ 0.085 microm. Plasma and lung samples were collected at 30, 60, 120, and 240 min postexposure. The concentrations of BaP parent compound and metabolites were determined by high-performance liquid chromatography. The toxicokinetic parameters were computed from the time course of plasma BaP concentration. The bioavailability of BaP increased as a function of exposure concentration, and toxicokinetic analysis indicates first-order pharmacokinetics for BaP. However, some toxicokinetic parameters such as clearance and volume of distribution remained constant throughout the duration of the postexposure period. BaP and its metabolite concentrations in plasma peaked at 1 h postexposure. At 240 min postexposure, only trace levels of BaP remained in the plasma. The BaP metabolites in the lung showed an identical trend where no parent compound was detected. Among the metabolites detected, BaP 4,5-, 7,8-, and 9,10-dihydrodiols, 3-OH-BaP, and 9-OH-BaP were predominant.

Comparative Metabolism, Bioavailability, and Toxicokinetics of Benzo[ a ]pyrene in Rats After Acute Oral, Inhalation, and Intravenous Administration

The metabolic fate of high doses of B a P is not fully established. To fill this important data need, a comprehensive metabolism, bioavailability, and toxicokinetic study has been undertaken to track the fate of B a P subsequent to single acute exposures. Doses of 100 mg/kg body weight, 0.1 mg/m 3 (equivalent to 19 mg/kg oral dose), and 4.5 w g/kg B a P were administered to 8-week-old male F-344 rats via oral, inhalation (nose only), and intravenous routes, respectively. Rats were sacrificed at 0, 0.5, 1, 2, 4, 6, 24, 48, and 72 hr postexposure. Blood, liver, lung, brain, reproductive tissues, urine, and feces samples were analyzed for parent B a P and metabolites by HPLC with fluorescence detection. Most of the administered B a P was metabolized 4, 6, and 72 hr postexposure for inhalation, intravenous, and oral routes, respectively. The following metabolites were detected: 4,5-dihydrodiol, 7,8-dihydrodiol, 9,10-dihydrodiol, 3,6-dione, 3-hydroxy, and 9-hydroxy B a P (organic fraction), glucuronides, sulfates, and glutathione conjugates (aqueous fraction). Toxicokinetic data revealed a high mean residence time, and low clearance values for B a P metabolites in lung, liver, and brain relative to plasma. Findings of this study establish the relationship between bioavailability and the acute toxic effects of B a P observed in our laboratory at these high doses.

Aryl hydrocarbon hydroxylase activity in F‐344 rats subchronically exposed to benzo(a)pyrene and fluoranthene through diet

In order to investigate the relationship between aryl hydrocarbon hydroxylase (AHH) activity and exposure to benzo[a]pyrene [B(a)p] and fluoranthene (FLA), AHH activities in liver tissues of male and female F‐344 rats were determined. Based on a range‐finding study, doses of 0, 5, 50, and 100 mg/kg B(a)p or 0, 150, 750, and 1500 mg/kg FLA were administered in the animal diet over a 90‐day period. After dosing, animals were sacrificed, liver tissues were removed, and microsomes were isolated. AHH activities were determined by reverse‐phase HPLC coupled with fluorescence detection using 3‐hydroxy B(a)p, and trans‐2,3‐dihydroxy‐1,10b‐epoxy‐1,2,3,10b tetrahydrofluoranthene as the standards. A dose‐dependent increase in enzyme activity was observed with increased B(a)p or FLA exposure in both males and females. Our results also demonstrate that B(a)p‐exposed females possess a higher AHH activity than males, but there is no significant sex difference with regard to enzyme activity in the case of FLA at higher doses. Overall, our findings suggest that long‐term exposure to the parent compound results in elevated levels of AHH activity, which may contribute to the formation of toxic reactive metabolites and subsequent symptoms in target organs.

Endocrine disruptive actions of inhaled benzo(a)pyrene on ovarian function and fetal survival in fisher F-344 adult rats.

This study evaluated the effect of inhaled BaP on female reproductive function. Rats were exposed to 50, or 75 or 100 μg BaP/m(3), 4 h a day for 14 days via inhalation. Plasma E(2), P(4), LH and FSH concentrations were determined. Ovarian BaP metabolism and aryl hydrocarbon hydrolase (AHH) activity at proestrus were determined and fertility evaluations were conducted. Ovulation rate and number of pups/litter were reduced in rats exposed to 100 μg BaP/m(3) compared with other treatment and control groups. Plasma concentrations of E(2), and LH were significantly reduced at proestrus in BaP-exposed versus those of controls whereas those of P(4) were significantly reduced at diestrus I. The activity of AHH in ovarian and liver tissues and concentrations of BaP 7,8-diol and BaP 3,6-dione metabolites increased in an exposure concentration-dependent manner. These data suggest that exposure of rats to BaP prior to mating contributes to reduced ovarian function and fetal survival.