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Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Repeated dosing with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively depletes small pre-antral follicles in the ovaries of rats and mice via apoptosis. The aryl hydrocarbon receptor (AhR) plays a role in mediating the effects of several xenobiotics. Therefore, this study was designed to investigate a potential role of the AhR in VCD-induced ovotoxicity. Female F344 rats, C57BL/6 mice, or AhR-deficient (-/-, AhRKO) mice were dosed daily (15 days) with vehicle, VCD (80 mg/kg, i.p.) and/or the AhR antagonist, alpha-naphthoflavone (ANF; 80 mg/kg, i.p.). Compared with controls, VCD caused a 60% reduction (P < 0.05) in primordial and primary follicles in mice and rats. Concurrent dosing with ANF protected against the VCD-induced follicle loss in rats, but not in mice. As with AhR-intact mice and rats, VCD induced a 70% loss (P < 0.05) of small pre-antral follicles in AhRKO mice. AhR mRNA expression was increased (P < 0.05) by VCD dosing in small pre-antral follicles isolated from ovaries of rats but not mice. AhR protein in rats was increased by VCD dosing in oocyte nuclei in primordial and primary follicles when measured by immunofluorescence and confocal microscopy. In rat small pre-antral follicles, apoptosis-associated caspase-3-like activity was increased (P < 0.05) by VCD treatment, decreased (P < 0.05) by ANF treatment, and unaffected by VCD plus ANF treatment. VCD had no effect on expression of GST Ya1 or GST Ya2 mRNA or CYP 1A1 protein in rats. Taken together, these findings demonstrate a difference between rats and mice in the potential involvement of AhR as related to VCD-induced ovotoxicity. Whereas, AhR appears to be involved in rats, no evidence for a similar role in mice was obtained. Overall, these findings point out that there can be mechanistic species differences in ovarian responses to xenobiotic chemicals.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and many of their toxic effects, including their immunotoxicities, are mediated by the activation of aryl hydrocarbon receptor (AhR). We previously reported that Aroclor 1254, one of the most widely used PCB mixtures, increased DNA fragmentation in mouse spleen cells, suggesting that apoptosis was correlated with the immunotoxicity of PCB (Yoo et al., Toxicol. Lett. 91, 83-89, 1997). In the present study we investigated the mechanism by which PCB induces apoptosis and the involvement of AhR in the PCB-mediated apoptosis of mouse spleen cells. Aroclor 1254 induced DNA fragmentation without AhR activation, and the apoptosis was unaffected by alpha-naphtoflavone, a well-known antagonist of AhR. Moreover, the PCB congeners (PCB 47, 52, 128, and 153), which have little affinity for AhR, induced DNA fragmentation, whereas congeners (PCB 77, 126, and 169) that have high affinity for AhR did not induce fragmentation. The di-ortho form of PCB (PCB 153) and Aroclor 1254 induced DNA fragmentation in the spleen cells of both AhR knockout mice and Ah low-response mice, whereas the non-ortho form of PCB (PCB 126) did not induce DNA fragmentation. In the light of these findings, it is evident that AhR is not involved in PCB-mediated apoptosis. PCB 153 significantly increased caspase-3 activity in both spleen cells and human leukemia cells, and z-VAD-fmk, a general inhibitor of caspases, prevented PCB-induced DNA fragmentation. Based on our findings, the most likely mechanism that can account for this biological effect involves the induction of caspase-dependent apoptotic cell death.

B cells have been identified as sensitive cellular targets responsible for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated suppression of humoral immunity. In previous studies, TCDD was shown to produce a significant inhibition of IgM secretion and mu gene expression in LPS-activated CH12.LX B cells (AhR expressing) but not in BCL-1 B cells (AhR deficient). The present studies extend these previous findings by investigating the effect of TCDD on AP-1 and nuclear factor (NF)-kappaB, both of which play an important role in B-cell activation, differentiation, and immunoglobulin (Ig) gene expression. Electrophoretic mobility shift assays and chloramphenicol acetyl transferase reporter gene experiments demonstrated that lipopolysaccharide (LPS)-induced DNA binding and transcriptional activity of AP-1 was markedly inhibited by TCDD at 24, 48, and 72 h after cellular activation of CH12.LX cells. Conversely, TCDD treatment produced no significant change on the activity of NF-kappaB. Two AhR antagonists, alpha-naphthoflavone and 2,2',5,5'-tetrachlorobiphenyl, attenuated TCDD-induced inhibition of AP-1 binding in CH12.LX cells. Concordant with this result, TCDD did not inhibit LPS-induced AP-1 activity in BCL-1 B cells. Moreover, supershift analysis revealed the major component of the AP-1 complex in LPS-activated CH12.LX cells was c-Jun. Additional studies revealed that the nuclear c-jun and c-jun steady-state mRNA expression was inhibited by TCDD treatment. Collectively, these results suggest that TCDD-induced inhibition of IgM expression by B cells may be mediated, at least in part, through a down-regulation of AP-1 activity in an AhR-dependent manner.

Nonmelanoma skin cancers represent the most common malignant neoplasms in humans. UV-B play a major role in the etiology of these tumors, but exposure to environmental procarcinogens is also involved. CYP catalyzes numerous chemical carcinogen bioactivations and effects of UV-B on their expression are poorly understood. The aim of this study was to explore the molecular events involved in the induction of CYP1B1, a major cutaneous CYP, by UV-B. Our results demonstrated that unique UV-B exposure (20 mJ/cm(2)) increases human CYP1B1 transcript in primary keratinocytes and HaCaT cell cultures. Among 20 human samples studied, we observed a large interindividual variability of CYP1B1 mRNA induction (1.1- to 4.5-fold). Pretreatment with an antioxidant, N-acetylcysteine, repressed CYP1B1 increase, suggesting the involvement of UV-B photoproducts. alpha-Amanitin inhibition studies and CAT assays demonstrated that CYP1B1 mRNA induction is associated with a transcriptional activation of its expression. alpha-Naphthoflavone inhibition studies and CAT assays performed after directed mutagenesis of xenobiotic responsive element sites showed the involvement of Ah receptor. Taken together, these data demonstrated that UV-B induces CYP1B1 gene expression after an activation of its transcription, which involves Ah receptor.

In the present study, 7-ethoxyresorufin O-deethylase (EROD), 7, 12-dimethylbenz[a]anthracene (DMBA)-hydroxylase, and covalent binding of (3)H-labeled 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole ((3)H-Trp-P-1) and (3)H-DMBA were examined in human umbilical vein endothelial cells (HUVEC) and human umbilical artery endothelial cells (HUAEC) exposed to the aryl hydrocarbon (Ah) receptor agonist beta-naphthoflavone (BNF) or vehicle only. The results revealed a marked induction of enzymatic activity in BNF-treated HUVEC compared with vehicle-treated cells, whereas no similar response was observed in BNF-treated HUAEC. EROD, DMBA hydroxylase, and covalent binding of (3)H-Trp-P-1 and (3)H-DMBA in BNF-treated HUVEC were reduced in the presence of the CYP1A inhibitor ellipticine. Addition of other CYP1A inhibitors alpha-naphthoflavone, miconazole, 1-ethynylpyrene, 1-(1-propynyl)pyrene), or the CYP1A substrate ethoxyresorufin to the incubation buffer of BNF-treated HUVEC reduced covalent binding of (3)H-Trp-P-1 by 93-98%. Western blot analysis confirmed an induction of CYP1A1 in BNF-treated HUVEC, but not in BNF-treated HUAEC. CYP1A1 was, however, detected in both vehicle- and BNF-treated HUAEC. The results showed that BNF exposure induced CYP1A1 and metabolic activation of xenobiotics in HUVEC, whereas the catalytic activity remained low in BNF-treated HUAEC. Our results suggest that endothelial lining of human veins may be a target for adverse effects of xenobiotics activated into reactive metabolites by Ah receptor-regulated enzymes. Several studies have detected CYP1A1 in endothelial linings, whereas expression of CYP1A2 and CYP1B1 seems to be negligible at this site. This suggests that the metabolic activation and covalent binding of (3)H-Trp-P-1 and (3)H-DMBA in HUVEC are most likely mediated by CYP1A1., (Copyright 2000 Academic Press.)

Winter flounder (Pleuronectes americanus) liver microsomes metabolized chlorzoxazone (CZX), a CYP2E1 (cytochrome P450 2E1)- specific substrate, to a single product, 6-OH CZX. The liver microsomal fraction had the highest level of activity, which varied among fish, ranging from 60 to 220 pmol/mg/min. CZX metabolism was linearly related to microsomal protein and dependent on NADPH. The optimal pH range for this activity was from 7.2 to 8.0. Maximal activity was found at 18-22 degreesC (154 +/- 62 pmol/mg/min) but was detectable at all temperatures tested. CZX 6-hydroxylation displayed first-order kinetics as shown by Lineweaver-Burk plots. The average apparent Km, 280 microM, was higher than that reported for human liver microsomes but similar to the Km determined for human CYP2E1 expressed in vaccinia virus. Vmax values ranged from 65 to 141 pmoles/mg/min. The CYP2E1-specific inhibitors, diethyldithiocarbamate (DDC) and aniline, significantly reduced 6-OH CZX production by 80 and 35%, respectively. 7,8-Benzoflavone and ethoxyresorufin, both CYP1A-specific inhibitors, did not significantly inhibit CZX metabolism nor did trioleandomycin (TAO) a CYP3A-specific inhibitor. Diethyldithiocarbamate (DDC), at 50 microM, had no significant effect on winter flounder liver microsomal ethoxyresorufin-O-deethylase activity (EROD), a CYP1A-specific activity. 7,8-Benzoflavone, at 10 microM, inhibited EROD activity by 60%. This is the first report of CYP2E1 activity in teleost liver microsomes., (Copyright 1998 Academic Press.)

Previous studies performed in this laboratory have shown that certain benzo(a)pyrene (BaP) metabolites, such as benzo(a)pyrene-7,8-dihydrodiol (BaP-7,8-diol) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), were more effective in elevating intracellular Ca2+ in normal human peripheral blood mononuclear cell (HPBMC) T and B cells than was BaP. Additionally, it has been shown that the suppression of human T cell mitogenesis produced by polycyclic aromatic hydrocarbons (PAHs) and certain BaP metabolites is reversed by treatment with alpha-naphthoflavone (ANF), a cytochrome P450 1A and 1B inhibitor. ANF also diminishes the elevation in intracellular calcium (Ca2+) produced by BaP in HPBMC. In the present studies, we further defined the relationships between intracellular Ca2+ elevation produced by BaP and two immunotoxic P450-derived metabolites, BaP-7,8-diol and BPDE in the Daudi human B cell line. At 1, 4, and 18 h, both BaP-7,8-diol and BPDE produced a significant rise in intracellular Ca2+. This effect, however, was not observed with BaP or benzo(e)pyrene (BeP), a nonimmunotoxic PAH. To evaluate the potential role of cytochrome P450 metabolism in PAH-induced Ca2+ elevation, Daudi cells were pretreated with ANF for 4 h, followed by treatment with BaP metabolites for 18 h. ANF completely reversed the rise in Ca2+ produced by BaP-7,8-diol, but had no effect on the Ca2+ elevation produced by BPDE. These results suggest that BPDE may be the ultimate P450 metabolite responsible for Ca2+ elevation in human B cells. BaP-7,8-diol and BPDE were found to increase tyrosine phosphorylation in Daudi whole cell lysates and to increase tyrosine phosphorylation of two important Src-related protein tyrosine kinases (PTKs), Lyn and Syk. Inhibition of tyrosine phosphorylation by herbimycin A was found to largely prevent the increase in intracellular Ca2+ produced by BaP-7,8-diol and BPDE, suggesting that Ca2+ elevation is coupled to increased tyrosine phosphorylation in Daudi. BPDE was found to produce a statistically significant increase in tyrosine phosphorylation of Lyn and Syk within 10 min of exposure. Collectively, these data demonstrate that certain P450-derived metabolites of BaP may be responsible for PTK activation and an increase intracellular Ca2+, which may alter antigen receptor signaling in human B cells.

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) alter Ca2+ homeostasis and inhibit activation of both B and T lymphocytes obtained from rodents and humans. In the present studies, we demonstrate that alpha-naphthoflavone (ANF), an inhibitor of cytochrome P4501A activity, reduced the Ca2+ elevation produced by BaP in human peripheral blood mononuclear cell (HPBMC) lymphocytes. These results suggested that BaP metabolites may play a role in intracellular Ca2+ homeostasis in human lymphocytes. Reactive oxidative intermediates of BaP produced in HPMBC are known to be highly carcinogenic and have also been shown to be immunosuppressive. We examined the effects of benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), benzo(e)pyrene (BeP), and anthracene, as well as certain BaP metabolites, on the levels of intracellular Ca2+ and glutathione in HPBMC. While BaP, DMBA, BeP, and anthracene did not cause a statistically significant decrease in GSH in HPBMC at concentrations of 1 or 10 microM following a 6-, 48-, or 72-hr exposure, reactive BaP metabolites including 4,5-epoxide BaP and 7,8-diol-9,10-epoxide BaP consistently produced a 20-30% depletion of glutathione in HPBMC following a 6-hr treatment period. These BaP metabolites also elevated intracellular Ca2+ in HPBMC during a 6-hr incubation. Results of these experiments suggest that metabolism of BaP to certain epoxide metabolites may be responsible for sulfhydryl damage leading to transient GSH depletion and Ca2+ elevation. These results are consistent with the hypothesis that sulfhydryl damage by certain PAH metabolites may lead to altered Ca2+ homeostasis, leading to inhibition of cell activation and proliferation in HPBMC.

In spite of increasing numbers of insecticides used in agriculture, there are serious concerns regarding the potential risks of exposure to these agents. Carbaryl is one of the most important carbamate insecticides and has been used for about 30 years to control a wide range of pests. The study was designed to investigate if, among various insecticides currently used in world agriculture, this compound could induce human CYP1A1, an enzyme known to play an important role in the chemical activation of xenobiotics to genotoxic derivatives. Studies on HepG2 and HaCaT cell lines showed that carbaryl is capable of increasing, in a dose-dependent manner, both the ethoxyresorufin rufin-O-dec, O-deethylase activity and the steady-state concentrations of CYP1A1 mRNA, suggesting a transcriptional activation of this gene. When alpha-naphthoflavone, a partial Ah receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element (XRE), were added to the cultures, CYP1A1 induction was suppressed. However, competitive binding studies using the 9S enriched fraction of human cytosol indicated that carbaryl did not displace [3H]TCDD from AhR. These data, together with the activation of a XRE-directed CAT reporter gene by carbaryl, suggest that induction of CYP1A1 involves the participation of the AhR and the XRE, but is not mediated by a direct carbaryl-receptor interaction. An alternative ligand-independent mechanism should be considered. Therefore, although carbaryl concentration in food is very low, care should be taken because of its possible adverse effects in human health through liver and skin, given the well established toxicological importance of CYP1A1 induction.

Cigarette smoke is known to induce cytochrome P4501A1 expression and activity in a variety of species. Although the elevation of this isozyme is assumed to be associated with the activation of the CYP1A1 gene through a ligand-mediated mechanism involving the Ah-receptor (AhR), this has not been determined. In this study we have examined the mechanism by which an ambient level of aged and diluted sidestream cigarette smoke (ADSS) induces cytochrome P4501A1. Effects of ADSS on C57BL/6N and DBA/ 2N mice were examined. Induction of P4501A1-associated ethoxyresorufin-O-dealkylase (EROD) activity was observed in the lungs of C57BL/6N mice, while there was no induction in DBA/ 2N mice. ADSS also induced EROD in wild-type mouse hepatoma (Hepa1c1c7) cells (hepa1), but not in variant hepa1 cells defective in the AhR nuclear translocator (ARNT) protein. ADSS exposure of recombinant hepa1 cells, stably transfected with a reporter plasmid containing the luciferase gene under control of several dioxin responsive enhancers (DREs), resulted in a time- and exposure-dependent induction of luciferase activity. ADSS-mediated induction of luciferase activity was inhibited by alpha-naphthoflavone (alpha NF), an Ah-receptor antagonist. Gel retardation analysis demonstrated that exposure to ADSS induced transformation and DNA binding of the AhR complex. In summary, our results not only indicate a role for the AhR in mediating the induction of P4501A1 by ADSS, but also demonstrate that environmentally relevant levels of ADSS must contain AhR ligands at sufficient concentrations to activate gene expression in an AhR-dependent manner.

The immunosuppressive effects of polycyclic aromatic hydrocarbons (PAHs) on immune responses in rodents, both in vivo and in vitro, have been widely documented. However, few studies have addressed the immunotoxicity of PAHs in the human system. In this report, we examined the toxic effects of nine different PAHs on human peripheral blood T cell mitogenesis. We found that benzo(a)pyrene (BaP), 3-methylcholanthrene (3-MC), and 7,12-dimethylbenz(a)anthracene (DMBA) were highly immunotoxic in the human system, while dibenz(a,c)anthracene (DAC) and dibenz(a,h)anthracene (DAH) were of intermediate toxicity, 9,10-dimethylanthracene (DMA), benzo(e)pyrene (BeP), and benz(a)anthracene (BA) were mildly immunotoxic, and anthracene (ANTH) had no measureable toxicity at the concentrations tested. Our results using human lymphocytes differed from previous studies in rodents, in that BaP and 3-MC were the most immunotoxic PAHs in the human mitogenesis assay, while DMBA has long been regarded as the PAH that is most potently toxic to rodent T cell responses. We also showed that alpha-naphthoflavone (ANF), which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 activity, was able to block the suppressive effects of both BaP and DMBA, but not 3-MC. This suggests that the immunotoxicity of 3-MC may be mediated through a different mechanism than BaP or DMBA. Addition of four different BaP metabolites directly to cultures of human mononuclear cells showed that the 7,8-dihydrodiol metabolite was the most toxic, and that this toxicity could be completely blocked by equimolar and 10-fold greater concentrations of ANF. The 7,8-dihydrodiol metabolite was probably further metabolized to the 7,8-diol epoxide, the toxicity of which could not be effectively reversed by ANF. The 4,5-epoxide metabolite was apparently cytotoxic at high concentrations (10 microM), while the 7-hydroxy metabolite had no overtly negative effects on proliferation.

Vitellogenesis or egg yolk production represents a key estrogen initiated process in oviparous vertebrates which is crucial for oocyte maturation. Previous in vitro studies have shown that cytochrome P4501A1 (CYP1A1)-inducing compounds such as beta-naphthoflavone (betaNF) modulate 17 beta-estradiol-induced vitellogenin (Vg) synthesis in primary cultures of juvenile rainbow trout liver cells. In this study, treatment of juvenile trout with 0.5 mg/kg 17 beta-estradiol plus either 25 or 50 mg/kg betaNF confirmed in vivo that betaNF may depress Vg synthesis by the liver. Alternatively, trout treated with 0.5 mg/kg 17 beta-estradiol plus 12.5 mg/kg betaNF or 5 mg/kg 17 beta-estradiol plus 12.5, 25, or 50 mg/kg betaNF showed a potentiation of Vg synthesis relative to estradiol-only injected fish. These results are significant as they suggest that changing plasma estrogen levels in sexually maturing trout will determine whether or not CYP1A1-inducing compounds may suppress, have no effect, or potentiate estrogen-induced liver Vg synthesis. Depressed Vg synthesis by trout injected with 0.5 mg/kg 17 beta-estradiol plus 50 mg/kg of betaNF correlated with depressed estrogen-binding capacity of liver, as assessed by [3H]17 beta-estradiol binding to liver nuclear protein extracts. Using gel mobility shift assay, the decrease in estrogen responsiveness of liver was not attributed to depressed estrogen response element-estrogen receptor binding. The fish liver vitellogenesis process offers a comparative model with which to further study the mechanism(s) of aryl hydrocarbon receptor-mediated antiestrogenicity and endocrine disruption.

In beta NF-induced channel catfish, hepatic ethoxyresorufin-O-deethylase (EROD) activity decreased 66.5% 24 hr after injection of 2-aminoanthracene (AA, 10 mg/kg) compared with non-AA-injected animals (p < 0.05). This difference in hepatic EROD activity was also significant 48 hr after treatment (p < 0.05), but no significant difference was observed after 4 or 7 days. Immunoblot analysis of hepatic microsomal protein from fish 24 hr after treatment with AA revealed two bands cross-reacting with CYP1A-specific monoclonal antibody 1-12-3: an apparently native CYP1A protein (52 kDa) and a 30-kDa protein. Furthermore, these two proteins were preferentially bound by [3H]AA compared with other microsomal proteins. Interestingly, the 30-kDa protein was observed only in fish exposed to AA and was immunoprecipitable with 1-12-3. In a separate in vivo experiment, hepatic EROD activity decreased and the 30-kDa protein increased with increased dose of AA. The 30-kDa protein is thought to be a CYP1A degradation product. In vitro experiments helped elucidate the mechanisms of interaction between AA and CYP1A. Incubation of microsomes with AA, prior to analysis of these microsomes for EROD activity, resulted in a NADPH- and time-dependent inhibition of EROD activity. Additionally, the P450 inhibitors 1-phenylimidazole and 3,3',4,4'-tetrachlorobiphenyl were used to decrease the binding of AA to CYP1A, suggesting that the binding of AA to CYP1A requires the enzymatic activity of CYP1A. It is proposed that mechanism-based inactivation of CYP1A by AA accounts for the observed AA-dependent decrease in hepatic EROD activity in vitro and in vivo in channel catfish.

The cellular localization of inducible CYP1A and constitutive CYP3A-like forms in different organ systems of Atlantic cod (Gadus morhua) was determined in control fish and fish exposed to beta-naphthoflavone (BNF). Paraffin-embedded sections were stained with polyclonal rabbit anti-cod P450 1A IgG or rabbit anti-rainbow trout P450con (a putative CYP3A form which cross-reacts with purified cod P450b) serum by the avidin-biotin peroxidase complex method. Following BNF-exposure of cod, CYP1A induction was immunohistochemically demonstrated in hepatocytes and endothelial cells of liver, the endocardium and vascular endothelium in the atrium and ventricle, and epithelial cells of the proximal tubular segment, endothelial cells, and interrenal cells in kidney. The vascular endothelium was the main site of induction of CYP1A in gills, spleen, gut, pyloric caecae, and gonads. The CYP3A-like isozyme P450b was mainly localized to hepatocytes, renal tubular epithelium, and epithelial cells of the mucosa in the intestine. Furthermore, the distribution of P450b was not affected by BNF exposure. The localization of P450b bears interesting similarities to the localization of CYP3A in mammals supporting the CYP3A-like identity of cod P450b. Simultaneous localization of inducible CYP1A and a constitutively expressed CYP isoenzyme has not previously been reported in fish. This is also the first presentation of cellular distribution of a CYP3-like isozyme in fish. Staining of CYP1A in endothelial cells supports previous observations that endothelium is a major site of CYP1A induction following xenobiotic exposure in fish. The observation of CYP1A induction in interrenal cells has important implications for possible endocrine effects of xenobiotic exposure.

The effect of phenobarbital (PB) and beta-naphthoflavone (beta-NF) on acetaminophen (APAP)-induced hepatotoxicity was evaluated in obese and lean Zucker rats. In addition, the consequences of APAP overdose on hepatic CYP2B1/2B2 enzyme activities following PB treatment were assessed. A single oral dose of APAP 3 g/kg (total body weight) was administered to both littermates 24 hr after the last dose of PB or beta-NF. Histologic evidence of hepatocellular necrosis and serum hepatic aminotransferase enzymes 48 hr after APAP administration was utilized to evaluate hepatic damage. Hepatic microsomal total cytochrome P450 concentrations, alkoxyresorufin O-dealkylase activities, and 16 beta-testosterone hydroxylase activities were determined to evaluate the effect of APAP overdose on cytochrome P450 enzyme-substrate activities in the presence and absence of PB treatment. APAP overdose produced lower hepatotoxicity in the obese Zucker rat compared to lean controls. A similar trend was observed in animals treated with PB prior to APAP administration. In contrast, beta-NF treatment produced potentiation of APAP toxicity and/or death of both obese and lean Zucker rats. Generally, APAP overdose produced reduction of hepatic cytochrome P450 enzyme-substrate activities. However, obese Zucker rats retained a higher percentage of their pre-APAP-treated enzyme activities which is consistent with the observation that obese Zucker rats are less affected by the hepatotoxic effects of APAP overdosage.

In rats, 1-nitronaphthalene (1-NN) causes both pulmonary and hepatic toxicity. Pulmonary toxicity is evident as bronchiolar damage, with necrosis of Clara cells and ciliated cells, whereas hepatic injury involves vacuolation of centrilobular hepatocytes. Pretreatment with O,O,S-trimethylphosphorodithioate [OOS-MeP(S)] or p-xylene gave three- to fourfold protection against 1-NN toxicity. These pretreatments also prevented both the increase in lung weight and the rise in gamma-glutamyltranspeptidase and alkaline phosphatase activity in bronchoalveolar lavage fluid normally associated with 1-NN toxicity. Pretreatment with Aroclor 1254 or beta-naphthoflavone (beta-NF) did not alter the LD50 of 1-NN. Aroclor or beta-NF pretreatment did, however, prevent morphological signs of lung injury and any increase in either lung weight or enzyme activity in bronchoalveolar lavage fluid. Liver damage was not prevented by these treatments; indeed, injury was exacerbated and was transferred from centrilobular to periportal areas. In control rats the covalent binding of [1-14C]NN to liver microsomes was eight times greater than to lung microsomes. Pretreatment with OOS-MeP(S) decreased covalent binding to lung microsomes, without affecting binding to liver microsomes. Conversely, both Aroclor and beta-NF slightly increased covalent binding in lung, but increased liver binding by 250-300%. Phenobarbitone also increased binding to liver microsomes by 250-300%, but failed to increase, or alter, the distribution of liver damage. The reported effects of these pretreatments indicate that the toxicity of 1-NN is probably activated by isoenzyme CYP2B1 in lung, but by isoenzymes CYP1A1 or CYP1A2 in the liver.

alpha-Naphthoflavone (alpha NF) and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [3H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10(-6) M alpha NF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10(-6) M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. alpha NF was further investigated as an Ah receptor antagonist by determining the inhibition by alpha NF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10(-9) M) inhibited the 17 beta-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, alpha NF (10(-8) to 10(-6) M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, alpha NF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. alpha NF (10(-6) M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17 beta-estradiol-induced responses in MCF-7 cells. Tamoxifen (10(-7) M), an antiestrogen which acts through the estrogen receptor, also inhibited 17 beta-estradiol-induced cell proliferation and alpha NF did not affect the tamoxifen-mediated antiproliferative response. Thus, alpha NF antagonized TCDD-induced CYP1A1 gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDD-mediated antiestrogenicity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10(-9) M TCDD plus alpha NF (10(-8) to 10(-6) M) or 10(-6) M alpha NF alone. Thus, alpha NF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10(-5) to 3 x 10(-5) M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10(-4) M) or clotrimazole (CTZ; 10(-5) M), or of 3A1 mRNA in cells cotreated with PB (2 x 10(-3) M), but not dexamethasone (10(-5) M. LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with beta-naphthoflavone (10(-5) M) or ciprofibrate (10(-4) M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 x 10(-4) to 3 x 10(-3) M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10(-6) to 10(-5) M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

The capacity for polycyclic aromatic hydrocarbons (PAH) to suppress immune cell function has been well documented. Nevertheless, mechanisms responsible for PAH immunosuppression and potential effects of PAH on lymphocyte development (lymphopoeisis) remain poorly defined. Murine bone marrow cultures were used in the present studies to determine if and by what mechanism(s) benzo[a]pyrene (B[a]P), a prototypic and highly carcinogenic PAH, suppresses B cell lymphopoiesis. Emphasis was placed on similarities between the processes leading to transformation and immunosuppression and on a possible role for programmed cell death (apoptosis) in B[a]P lymphotoxicity. Data presented herein indicate that: (1) B[a]P suppresses B cell lymphopoiesis in bone marrow cultures at extremely low concentrations (10(-8) M); (2) benzo[e]pyrene, the relatively noncarcinogenic congener of B[a]P, is approximately 1000 times less potent than B[a]P in suppressing B cell lymphopoiesis; (3) bone marrow cells from PAH-resistant DBA/2 mice are less sensitive to B[a]P than cells from C57BL/6 mice; (4) B[a]P induces preB cell apoptosis; and (5) alpha-naphthaflavone, an inhibitor of Ah-receptor dependent, P450 isoenzyme activity, blocks B[a]P-mediated preB cell apoptosis and inhibits B[a]P-dependent suppression of lymphopoiesis. The results support the hypothesis that B[a]P suppression of B cell lymphopoiesis is mediated at least in part by the induction of programmed cell death and that the Ah receptor and/or P450 isoenzymes are involved in this process. The results suggest the potential for PAH to affect development of the B lymphocyte repertoire.

Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10(-9) to 10(-5) M. DMBA and benzo[a]pyrene (B[a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to splenocyte cultures produced a concentration-dependent suppression of the PFC response, in which B[a]P was found to be 17.5-fold more potent than DMBA. These results are in contrast to those found in vivo, where DMBA has been shown to be more potent than B[a]P at suppressing humoral immunity. The 3,4-diol metabolite of DMBA produced a concentration-dependent suppression (10(-8) to 10(-5) M) of the in vitro PFC response and was found to be 65-fold more potent than the parent compound DMBA. In contrast, the 5,6-diol metabolite of DMBA had no effect on the PFC response or cell viability. Both the 3-OH-DMBA and 7-hydroxymethyl-12-methyl-benz[a]anthracene (7-OHMe-12-Me-BA) metabolites were found to be immunosuppressive at concentrations of 10(-6)M. Furthermore, suppression by 7-OHMe-12-Me-BA was observed at concentrations as low as 10(-8) M. Immunosuppression by the 7-Me-12-OHMe-BA and the di-OHMe-BA metabolites was only observed at high (10(-5) M) concentrations. The cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), was utilized to determine if cytochrome P450-mediated metabolism is involved in DMBA-induced suppression of the in vitro PFC response. ANF (10(-5) M) reversed the DMBA-induced immunosuppression seen at 10(-5) M and attenuated the immunosuppression at 3 x 10(-5) and 10(-4) M. The results of these studies demonstrate that several metabolites of DMBA which can be generated by the cytochrome P450-dependent monooxygenase systems are immunosuppressive in the in vitro PFC response assay. Furthermore, the cytochrome P450 inhibitor, ANF, was able to reverse DMBA-induced immunosuppression.(ABSTRACT TRUNCATED AT 400 WORDS)

Based on our previous observations (H. S. Ramsdell and D. L. Eaton, 1990, Cancer Res. 50, 615-620) that the proportion of aflatoxin B1 (AFB1) converted to the highly reactive AFB1-8,9-epoxide in microsomal incubations varies with substrate concentration, we have examined the hypothesis of T. Shimada and F. P. Guengerich (1989, Proc. Natl. Acad. Sci. USA 86, 462-465) that cytochrome P450 IIIA4 is principally responsible for the activation (epoxidation) of AFB1 by human liver microsomes. The initial rates of formation of AFB1-8,9-epoxide and hydroxylated AFB1 metabolites were determined in microsomes prepared from livers of organ donors (n = 14) at AFB1 concentrations of 124 and 16 microM. Microsomal oxidation of nifedipine, catalyzed primarily by P450 IIIA enzymes, was also determined by HPLC. Rates of formation of AFB1 metabolites and nifedipine oxidation were poorly correlated at either AFB1 concentration (r2 = 0.13-0.41). A somewhat better correlation between AFB1 epoxidation and nifedipine oxidation was observed at 124 microM AFB1 (r2 = 0.41) than at 16 microM AFB1 (r2 = 0.26). Treatment of pooled microsomes with troleandomycin, an apparently specific inhibitor of P450 IIIA enzymes, resulted in 35% inhibition of AFB1-8,9-epoxide formation at the high AFB1 level but had little effect at 16 microM AFB1. An antibody against rat cytochrome P450 IIIA1 significantly inhibited AFB1 epoxidation at high, but not low, AFB1 concentrations, whereas AFQ1 formation was strongly inhibited at all substrate levels examined. These results are consistent with the hypothesis that cytochrome P450 IIIA enzyme(s) can form AFB1-8,9-epoxide, but are effective at only relatively high substrate concentrations. Another P450 enzyme(s) appears to be principally responsible for AFB1-8,9-epoxide formation at the low AFB1 levels that would be typical for dietary exposures.

Preliminary investigations into the role of biotransformation in 1,2,3-trichloropropane (TCP)-induced tumor formation have been undertaken. Male F-344 rats were administered 30 mg/kg [14C]TCP (100 microCi/kg) ip and killed 4 hr later. The extent of covalent binding to hepatic protein, DNA, and RNA was 418, 244, and 432 pmol [14C]TCP equivalents/mg, respectively. An in vivo covalent binding time course showed no significant change in [14C]TCP equivalents bound to hepatic DNA (1-48 hr), while binding to protein was maximal by 4 hr and decreased significantly by 48 hr. The binding of TCP-associated radioactivity to hepatic protein and DNA was shown to be cumulative for two and three doses when given 24 hr apart. Pretreatment of animals with phenobarbital caused a decrease while pretreatment with SKF 525-A caused an increase in covalent binding of [14C]TCP equivalents to protein and DNA. Pretreatment of rats with beta-naphthoflavone did not alter the covalent binding of [14C]TCP equivalents to protein or DNA. However, glutathione depletion with L-buthionine-(R,S)-sulfoximine increased binding to protein by 342% while it decreased binding to DNA by 56%. Intraperitoneal administration of TCP also depleted hepatic GSH by 41 and 61% 2 hr after doses of 30 and 100 mg/kg. The in vivo binding data suggest a dual role for GSH in the bioactivation of TCP. It may, in part, be that GSH is involved in the bioactivation and covalent binding of TCP to hepatic DNA. However, it also appears to detoxify a reactive intermediate(s) that binds to protein.

The interaction between beta-naphthoflavone induction (BNF: 100 mg/kg) and carbon tetrachloride (CCl4; 1 ml/kg) hepatotoxicity was examined in the flounder. Treatment groups composed of control, BNF, CCl4, and BNF/CCl4 were compared in terms of cytochrome P450 isozyme content (LM4b; LM2), catalytic activity, isozyme distribution. SGOT-SGPT levels, and pathology. CCl4 administration resulted in significant reductions in both the constitutive P450 (LM2) and the BNF-inducible isozyme (LM4b) as well as elevations in SGPT and SGOT levels. The decline in LM4b isozyme content was reflected by stoichiometric decreases in ethoxyresorufin-O-deethylase activities. BNF/CCl4 coadministration was protective in part against CCl4 hepatotoxicity. Immunohistochemistry indicated that LM4b was diffusely distributed throughout the liver. These interactions have demonstrated a multiple P450 isozyme involvement, the protective nature of BNF against CCl4 hepatotoxicity in the flounder, the ability to maintain an inductive response in face of CCl4 coadministration, and the diffuse distributional pattern of LM4b in the flounder liver.

The inhibition of cerebral cortex and medulla oblongata acetylcholinesterase and hepatic and plasma aliesterases was studied in female rats following intraperitoneal administration of the phosphorothionate insecticide parathion or its active metabolite paraoxon. Acetylcholinesterase is the target enzyme for organophosphate toxicity while aliesterases are alternative targets for organophosphate inhibition which could serve in a protective capacity during organophosphate intoxication. The effects of pretreatment with cytochrome P450 inducers and inhibitors were also investigated. Pretreatment with phenobarbital slowed the time course of acetylcholinesterase and hepatic aliesterase inhibition following parathion exposure, suggesting the induction of a detoxication pathway(s) to a greater extent than the induction of activation. Although pretreatment with piperonyl butoxide did not affect the rate of acetylcholinesterase inhibition, it slowed hepatic and plasma aliesterase inhibition following parathion administration, presumably from inhibition of the parathion activation pathway. In rats pretreated with beta-naphthoflavone (BNF), hepatic aliesterases demonstrated lower specific activity; additionally, there was a reduced level of inhibition in BNF-pretreated rats following either parathion or paraoxon administration. However, any effects of BNF on parathion- or paraoxon-induced inhibition cannot be distinguished at this time from the effects of the oil vehicle, which reduced esterase inhibition, presumably by its ability to sequester the organophosphate. Brain acetylcholinesterase was partially inhibited before the hepatic aliesterases were maximally inhibited in all treatment groups. In most cases, plasma aliesterases were maximally inhibited within 15 min after organophosphate administration. Thus hepatic aliesterases constitute an important but not completely effective form of protection from the inhibitory effects of organophosphates.

The effect of treatment of pregnant C57BL/6J (B6) and AKR/J (AKR) mice with the cytochrome P-450 inducing agent beta-naphthoflavone (beta NF) on caffeine metabolism and on exposure of dam and embryo to caffeine and to its metabolites theophylline (TP), theobromine (TB), paraxanthine (PX), and trimethyluric acid (TMUA) was investigated. Treated dams were given either 20 or 80 mg beta NF/kg ip on Days 9 and 10 of gestation. Caffeine, 175 mg/kg, was given ip on Days 11 and 12 of gestation. Concentrations of caffeine and its metabolites were monitored in maternal plasma and in embryo homogenates at 0.083, 0.25, 0.5, 1, 2, 4, and 6 hr after the second caffeine injection. Effective exposure was expressed as the area under the concentration-time curve from 0 to 6 hr. Caffeine loss in B6 mice was best described by an expression incorporating capacity-limited kinetics. Pretreatment induced formation of TB and PX, while it induced elimination of all three of the monodemethylated metabolites, in B6 mice. Consequently, pretreatment caused marked reductions in exposure of B6 dams and embryos to both caffeine and TP. Exposure to TB and PX was less consistently affected, although it was reduced by pretreatment with 80 mg beta NF/kg. In contrast, exposure of AKR dams and embryos to caffeine and its metabolites was only slightly affected by beta NF pretreatment. The prevalence of paw malformations in the offspring of treated mice correlated well with effective exposure to both caffeine and TP, but not at all with the administered caffeine dose.

Male and female CD-1 mice received single oral doses of benzene (220, 440, and 880 mg/kg) and were pretreated with modifiers of the mixed-function oxidase enzyme activities. Urinary metabolites (MT) (0-24 and 24-48 hr) were quantified by high-performance liquid chromatography. The micronucleus test was performed at 30 h. The following pretreatments were used to correlate micronucleus formation and the excreted benzene MT: 3-Methylcholanthrene and beta-naphthoflavone led to a marked increase in micronuclei (MN) and MT, whereas phenobarbital caused a slight increase, and SKF-525A had no effect. MN and MT were decreased when benzene was administered by the ip route or toluene was given simultaneously. Females had a lower number of MN and excreted more unconjugated phenol than did males. Muconic acid, hydroquinone, and phenol glucuronide and MN correlated well. They were dependent on both the dose and route of administration of benzene, being most inducible by P-448 inducers, in males more than females. The administration of hydroquinone induced MN, but phenol or catechol (200, 250, and 150 mg/kg, po, respectively) did not, and none of these compounds yielded trans, trans-muconic acid, a benzene MT in urine. This study establishes that benzene myeloclastogenicity is a function of its metabolism and that quantification of urinary metabolites could provide reliable correlates of this effect in vivo.

The time course of induction of the rainbow trout microsomal hepatic monooxygenase (MO) system was examined by determination of levels of mRNA and corresponding levels of catalytic activity. Animals were pretreated with beta-naphthoflavone (beta-NF, ip, 100 mg/kg) and terminated at 0, 2, 6, 18, and 48 hr postinjection. Levels of mRNA were determined by immunoprecipitation of in vitro translation products. Levels of mRNA coding for the cytochrome P-450 LM4b isozyme were maximally increased (13-fold) at 18 hr and had decreased almost to pretreatment levels by 48 hr post-treatment. This was in contrast to the catalytic activity in which ethoxyresorufin-O-deethylase (EROD) and ethoxycoumarin-O-deethylase (ECOD) were significantly elevated at both 18 hr (25- and 5-fold, respectively) and 48 hr (46- and 8-fold, respectively). Pretreatment with beta-NF (ip, 100 mg/kg) or 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB, ip, 150 mg/kg) for 18 hr resulted in significant differences in levels of mRNA in only the beta-NF-treated group. The LM2 P-450 isozyme could not be detected by immunoprecipitation with anti-LM2 IgG in trout treated with these same inducers. The results suggest a difference between the time course of induction of the mRNA for cytochrome P-450 LM4b isozyme and the induction of catalytic activity. Under the detection system utilized, the results suggest that the phenobarbital-like inducer, 6-CB, does not induce cytochrome activity nor does it induce the mRNA for cytochrome P-450 LM4b isozyme.

The effect of intraovarian (io) injection of benzo(a)pyrene (BP) on primordial oocyte number, ovarian aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.1) activity, and hepatic AHH activity and P-450 content was investigated in C57BL/6N (B6) and DBA/2N (D2) mice. Ovaries were exteriorized through dorsolumbar incisions under ether anesthesia, and 1.0 microliters of corn oil or corn oil containing BP was injected directly into the ovary. Bilateral io injection of BP was ovotoxic in a time and dose dependent manner. Primordial oocyte destruction was maximal 8 days after injection in both B6 and D2 mice. Unilateral io injection of BP destroyed oocytes only in the treated ovary. The threshold dose for primordial oocyte destruction was similar in both strains, approximately 50 ng/ovary. However, ED50's were different; 1.1 micrograms/ovary in B6 and 8.9 micrograms/ovary in D2 mice. Intraperitoneal treatment with alpha-naphthoflavone (80 mg/kg) inhibited the oocyte destruction by io treatment with BP (10 micrograms/ovary) in both B6 and D2 mice. In B6 mice, ovarian AHH activity was induced by BP in the treated ovary after either bilateral, or unilateral io treatment. However, io BP treatment did not change ovarian AHH activity in D2 mice. Hepatic AHH activity and P-450 content was not altered by io BP treatment in either mouse strain. These results strengthen the hypothesis that the ovary has the capability of metabolizing BP to ovotoxic products.

The effects of the cytochrome P450 inducer beta-naphthoflavone (BNF) on NO2 toxicity were studied in two strains of mice. In one strain (C57B1/6J), cytochrome P450 could be induced by the aromatic hydrocarbon, while in the other strain (DBA/2J) cytochrome P450 was not inducible by this compound. Mice were treated with BNF before and during 4 days of exposure to 20 ppm NO2. The body growth of NO2-exposed mice improved only in BNF-treated C57B1/6J mice. In this strain, BNF reduced both pulmonary edema (as measured by wet and dry lung weights or as assessed by histological studies) and lung peroxidation (as measured by malondialdehyde). This protective effect of BNF on NO2 toxicity in C57B1/6J mice was associated with an increase in the components of the cytochrome P450 system (cytochrome P450 and cytochrome b5), whereas the activities of pulmonary antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) were not significantly increased. These data suggest that the induction of the cytochrome P450 system may be important in promoting NO2 tolerance in those strains of mice in which the cytochrome P450 system is genetically inducible.

The potential role of the cytochromes P-450 in methylcholanthrene (MC)-mediated suppression of cutaneous delayed hypersensitivity (CDH) in C57BL/6J (B6) mice was evaluated indirectly by treating mice with agents known to induce or inhibit hepatic cytochromes P-450 prior to contact sensitization. Subsequent alterations in aryl hydrocarbon hydroxylase (AHH) activity and CDH, as measured by suppression of 2,4-dinitrofluorobenzene (DNFB)-induced ear swelling, were measured. MC administration resulted in a dose-dependent suppression of ear swelling and a concomitant dose-dependent induction of hepatic AHH activity. Treatment of B6 mice with phenobarbital (PB), 80 mg/kg daily X 3, a broad spectrum inducer of P-450, resulted in a 2.5-fold increase in benzo[a]pyrene (B[a]P) hydroxylase activity without affecting CDH. Animals treated with the same PB protocol prior to an ED20 dose of MC showed no difference in suppression of CDH compared to animals treated with MC alone. In contrast, successive treatment with the selective P1-450 inducer, 5,6-benzoflavone (beta NF), prior to and following an ED20 dose of MC significantly increased suppression of CDH (p less than 0.001) usually seen at this MC dose. Treatment with a known inhibitor of cytochrome P1-450 activity, 7,8-benzoflavone (alpha NF), did not prevent AHH induction when administered prior to and following MC (ED100) nor did it suppress CDH when administered alone. However, this alpha NF treatment completely prevented suppression of CDH usually seen at this MC dose. These data provide evidence suggesting that metabolic activation by cytochrome P1-450 is required for the expression of the immunosuppressive activity of MC.

Treatment of goldfish with beta-naphthoflavone (ip) resulted in induction of hepatic monooxygenase activity in a dose-dependent manner. Levels of 7-ethoxyresorufin-O-deethylase and mexacarbate NADPH-dependent oxidative metabolism as well as cytochrome(s) P-450 were increased. Other substrates which have been associated with 3-methylcholanthrene (3-MC)-type induction in mammals were unchanged as was delta-aminolevulinic acid synthetase. Enzyme activities were inhibited by carbon monoxide and piperonyl butoxide confirming their cytochrome P-450 nature. NADH was found to stimulate metabolism in a significant manner with NADPH. pH-dependent ethyl isocyanide binding characteristics were similar in control and induced preparations while mexacarbate binding to oxidized microsomes revealed a higher apparent affinity constant (Kapps) as well as greater overall binding in induced preparations. SDS-gel electrophoresis revealed an increased band with a molecular weight of approximately 56,000.

The apparent (app) Km and app Vmax for mouse hepatic microsomal oxidative detoxification of chlorpyrifos to 3,5,6-tricholoro-2-pyridinol were 16.10 +/- 6.8 microM and 263.2 +/- 22.5 nmol/liver/min, respectively. The app Km and app Vmax for the oxidative activation of chlorpyrifos to chlorpyrifos oxon were 20.0 +/- 6.5 microM and 126.1 +/- 14.6 nmol/liver/min, respectively, whereas the app Km and app Vmax for hepatic microsomal hydrolysis of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol were 1.87 +/- 0.36 mM and 89,450.7 +/- 12,087.3 nmol/liver/min, respectively. Under first-order conditions the capacity of mouse hepatic microsomes to detoxify chlorpyrifos oxon exceeded their capacity to generate this potent cholinesterase inhibitor from chlorpyrifos by a factor of 7.6. Pretreatment of mice with phenobarbital (70 mg/kg daily for 4 days) resulted in a 2.5-fold increase in the app Vmax's for oxidative activation and detoxification of chlorpyrifos, and a 1.6-fold increase in the app Vmax for hydrolysis of chlorpyrifos oxon. The app Km's were not altered by phenobarbital pretreatment. Administration of beta-naphthoflavone (80 mg/kg/daily for 2 days) to mice resulted in a slight decrease in the app Vmax's for oxidative activation and detoxification of chlorpyrifos, without altering the app Km's of the same reactions, or the hydrolysis of chlorpyrifos oxon. Phenobarbital and beta-naphthoflavone increased and decreased, respectively, the predicted hepatic clearance of chlorpyrifos. The acute toxicity of chlorpyrifos was slightly antagonized by phenobarbital pretreatment, but was potentiated by beta-naphthoflavone administration. These pretreatments did not affect cholinesterase, nonspecific esterase, or plasma A-esterase activities. Collectively, these results suggest that chlorpyrifos oxon formed within the liver does not escape hydrolysis by the liver, and that extrahepatic sites of activation are important in directly mediating the acute toxicity of chlorpyrifos.

Groups of Atlantic cod and rainbow trout were treated (ip) with beta-naphthoflavone (BNF), phenobarbital, or peanut oil (controls), and properties of the hepatic xenobiotic and steroid metabolizing enzyme systems were evaluated. In both species, BNF treatment resulted in significant induction of microsomal 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase, biphenyl 4-hydroxylase, and phenanthrene oxidation, especially at the 1,2-position. Immunochemical studies with rabbit IgG prepared against the major BNF-inducible cytochrome P-450 in cod, P-450c, revealed increased amounts of immunoreactive protein in liver slices from both species after BNF treatment. The molecular weight of the induced protein was approximately 58,000 Da, as shown by Western blotting. When titrating biphenyl 4-hydroxylation, however, the antibodies distinguished between the two species, inhibiting the activity of BNF-induced cod 90% and that of rainbow trout 40% at 10 mg IgG/nmol P-450. Furthermore, cytochrome b5 content and UDP-glucuronyltransferase activity were significantly induced only in rainbow trout, whereas the specific content of cytochrome P-450 was significantly increased only in cod. Differences between the two species were observed in the levels of constitutive activities, the amount of induction, and in the regioselectivity of phenanthrene oxidation and androstenedione metabolism. Treatment with phenobarbital showed no effect on any of the parameters investigated in either species. The results show that although there are many common features of the hepatic xenobiotic and steroid biotransformation systems of the two teleosts, certain species characteristics exist in constitutive properties and induction responses.

Recent reports have shown that alpha-naphthoflavone (alpha-NF) in vivo enhances the sister chromatid exchange (SCE) frequency in lymphocytes from human populations exposed to cigarette smoke or polychlorinated biphenyls and dibenzofurans. In this study, female Sprague-Dawley rats (9-11 weeks old) were administered a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and killed 6 days after treatment. Blood cultures were established with or without alpha-NF. The baseline and alpha-NF-induced SCE frequencies were assessed in lymphocytes after a 72-hr culture period. No effect on the SCE baseline frequency (cultures without alpha-NF) was detected in rats exposed to 0-30 micrograms TCDD/kg. However, the SCE frequencies from cultures incubated in the presence of alpha-NF were significantly higher in lymphocytes from rats treated with TCDD. Moreover, delta SCE values (SCE alpha-NF minus SCE baseline) were significantly higher in lymphocytes from rats treated with TCDD than in controls. A dose-dependent increase in delta SCE values was observed between 0 and 3 micrograms TCDD/kg, followed by a plateau at higher doses. This induction pattern closely resembled the induction of the liver microsomal aryl hydrocarbon hydroxylase activity by TCDD. In contrast to TCDD, phenobarbital treatment of rats (75 mg/kg/day) had no effect on alpha-NF-induced SCE frequencies in lymphocytes. Liver microsomes from TCDD-treated rats metabolized alpha-NF at a rate much faster than that of control microsomes. These studies indicate that TCDD-exposed rats provide a useful model to investigate the mechanism of enhanced in vitro induction of SCE frequency in lymphocytes from humans exposed to toxic halogenated aromatics or cigarette smoke.

Rainbow trout were fed a diet containing indole-3-carbinol (2000 ppm), beta-naphthoflavone (500 ppm), or Aroclor 1254 (100 ppm) for 6 weeks before a single 24-hr exposure to an aqueous solution of 250 ppm diethylnitrosamine (DEN). The fish were killed 42 weeks later to determine the carcinogenic response. DEN exposure produced an 80.2% incidence of liver tumors and an average of 3.47 tumors per tumor-bearing fish, whereas no tumors were detected in the sham-treated control fish. Tumor induction was inhibited by indole-3-carbinol (27.5% incidence, 1.89 tumors per tumor-bearing fish) but enhanced by beta-naphthoflavone (91.8% incidence, 3.60 tumors per tumor-bearing fish). Aroclor 1254 had no effect on DEN-induced hepatocarcinogenesis (80.0% incidence, 3.03 tumors per tumor-bearing fish). The effects of these modulators on O6-ethylguanine and 7-ethylguanine formation (measured by HPLC and fluorescence spectrophotometry) were examined. Liver DNA ethylguanine levels were reduced in indole-3-carbinol-pretreated fish and increased in beta-naphthoflavone-pretreated fish compared to untreated controls after DEN exposure. Aroclor 1254 pretreatment had no significant effect on DNA ethylguanine formation. Similar O6-ethylguanine to 7-ethylguanine ratios were found among the control and treated groups. The results of this study indicate that modulation of DEN hepatocarcinogenesis by indole-3-carbinol and beta-naphthoflavone may be mediated by their effects on O6-ethylguanine formation and, therefore, on the initiation phase of carcinogenesis.

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.

Changes in cytochromes P-450, aminopyrine N-demethylase (APDM), aromatic hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), and type III spectral complex formation were measured in hepatic microsomes of control, phenobarbital (PB)-, and beta-naphthoflavone (beta NF)-induced rats after a single dose of dihydrosafrole (4-n-propyl-1,2-methylenedioxybenzene, DHS). Time profiles of changes in these microsomal parameters were complex and showed that APDM activities and cytochrome P-450 levels decreased immediately after treatment and were associated with concurrent increases in the intensity of the type III methylenedioxyphenyl (MDP) metabolite/cytochrome P-450 spectral complex. In noninduced rats, both APDM activity and cytochrome P-450 levels returned to control levels between 12 and 24 hr after treatment with DHS and subsequently increased above control levels. In PB- and beta NF-induced animals, the inhibitory phases were more prolonged and activity never returned to levels higher than the corresponding controls. AHH activity was increased substantially (two- to three-fold) in all cases after DHS administration. Although displacement of the MDP metabolite/cytochrome P-450 complex with 2-methylbenzimidazole generally led to a marked restoration of cytochrome P-450 levels and partially reversed the inhibition of APDM, it had little or no effect on AHH activities.

The response of hepatic microsomal cytochrome P-450 monooxygenase and UDPglucuronosyltransferase to a common inducer, beta-naphthoflavone, was investigated in rainbow trout acclimated to 5 or 17 degrees C. The hepatic microsomal 7-ethoxycoumarin-O-deethylase, 7-ethoxyresorufin-O-deethylase, and benzo[a]pyrene hydroxylase activities, measured at 5 or 17 degrees C, were induced at both acclimation temperatures. Maximum response of 7-ethoxyresorufin-O-deethylase and 7-ethoxycoumarin-O-deethylase activities to beta-naphthoflavone was obtained with doses of 5 mg/kg and above, both in warm- and cold-acclimated trout. In warm-acclimated trout the maximum degree of induction of cytochrome P-450-dependent activities was already evident on the day following treatment with beta-naphthoflavone (100 mg/kg), whereas these activities reached maximum values after 3 days in cold-acclimated fish. Furthermore, the maximum induced values of 7-ethoxyresorufin-O-deethylase and benzo[a]pyrene hydroxylase activities were higher in cold-acclimated than in warm-acclimated fish. beta-Naphthoflavone treatment caused a slight but significant increase (1.6-fold) in UDPglucuronosyltransferase activity in warm-acclimated fish when measured at 5 or 17 degrees C, whereas in cold-acclimated fish a significant elevation was seen only when the activity was determined at environmental temperature (5 degrees C).