Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.     

Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision‐cut rat liver slices

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision‐cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O‐deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O‐deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1‐methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1‐methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene‐mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764–775, 2017.     

The naturally occurring aliphatic isothiocyanates sulforaphane and erucin are weak agonists but potent non-competitive antagonists of the aryl hydrocarbon receptor

As the Ah receptor target gene products play a critical role in chemical carcinogenesis, antagonists are considered as potential chemopreventive agents. It is demonstrated in this paper that the isothiocyanates R,S-sulforaphane and erucin are non-competitive antagonists of the aryl hydrocarbon (Ah) receptor. Both isothiocyanates were poor agonists for the receptor and elevated CYP1A1 mRNA levels only modestly when incubated with precision-cut rat liver slices. In contrast, the classical Ah receptor agonist benzo[a]pyrene was a potent inducer of CYP1A1 mRNA levels, with this effect being effectively antagonized by the two isothiocyanates. In further studies, it was demonstrated that R,S-sulforaphane could both prevent the interaction of and displace already bound benzo[a]pyrene from the Ah receptor, but no concentration dependency was observed with respect to the isothiocyanate. Both erucin and R,S-sulforaphane antagonized the benzo[a]pyrene-mediated increase in the CYP1A-mediated O-deethylation of ethoxyresorufin in rat precision-cut liver slices. Of the two isomers of R,S-sulforaphane, the naturally occurring R-isomer was more effective than the S-isomer in antagonizing the activation of the Ah receptor by benzo[a]pyrene. Antagonism of the Ah receptor may be a major contributor to the established chemoprevention of aliphatic isothiocyanates.     

Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices.

The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 microM, respectively, with corresponding V(max) values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction.     

Dose-response studies on the induction of liver cytochromes P4501A1 and 1B1 by polycyclic aromatic hydrocarbons in arylhydrocarbon-responsive C57BL/6J mice

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,3',4'-tetrachlorobiphenyl, the dose-response studies of the induction of CYP1-dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresorufin, 7-ethoxycoumarin, (+/-)-benzo[a]pyrene-7,8-diol, dibenzo[a, pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresorufin O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[j]-fluoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,3',4'-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresorufin O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a,l]pyrene, dibenz[a,j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresorufin O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.     

Dose-response studies on the induction of liver cytochromes P4501A1 and 1B1 by polycyclic aromatic hydrocarbons in arylhydrocarbon-responsive C57BL/6J mice

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,30,40-tetrachlorobiphenyl, the dose–response studies of the induction of CYP1- dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresoru.n, 7-ethoxycoumarin, (±)-benzo[a]pyrene- 7,8-diol, dibenzo[a,l] pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresoru.n O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k].uoranthene, benzo[b].uoranthene, benzo [j] -. uoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,30,40-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresoru.n O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a, l] pyrene, dibenz[a, j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresoru.n O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.     

Tissue specific induction of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver and lung following in vitro (tissue slice) and in vivo exposure to benzo(a)pyrene.

Cytochrome P-450s (CYPs) detoxify a wide variety of xenobiotics and environmental contaminants, but can also bioactivate carcinogenic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), to DNA-reactive species. The primary CYPs involved in the metabolism and bioactivation of BaP are CYP1A1 and CYP1B1. Furthermore, BaP can induce expression of CYP1A1 and CYP1B1 via the aryl hydrocarbon receptor. Induction of CYP1A1 and CYP1B1 by BaP in target (lung) and non-target (liver) tissues was investigated utilizing precision-cut rat liver and lung slices exposed to BaP in vitro. Tissue slices were also prepared from rats pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce expression of CYP1A1 and CYP1B1. In addition, in vivo exposure studies were performed with BaP to characterize and validate the use of the in vitro tissue slice model. In vitro exposure of liver and lung slices to BaP resulted in a concentration-dependent increase in CYP1A1 and CYP1B1 mRNA and protein levels, which correlated directly with the exposure-related increase in BaP-DNA adduct levels observed previously in the tissue slices [Harrigan, J.A., Vezina, C.M., McGarrigle, B.P., Ersing, N., Box, H.C., Maccubbin, A.E., Olson, J.R., 2004. DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo(a)pyrene. Toxicological Sciences 77, 307-314]. Pretreatment of animals in vivo with TCDD produced a marked induction of CYP1A1 and CYP1B1 expression in the tissue slices, which was similar to the levels of CYP1A1 and CYP1B1 mRNA achieved in liver and lung following in vivo treatment with BaP. Following in vitro exposure to BaP, the levels of CYP1A1 were greater in the lung than the liver, while following all exposures (in vitro and in vivo), the levels of CYP1B1 mRNA were greater in lung tissue compared to liver. The higher expression of CYP1A1 and CYP1B1 in the lung was associated with higher levels of BaP-DNA adducts in the lung slices (Harrigan et al.'s work) and together, these results may contribute to the tissue specificity of BaP-mediated carcinogenesis.     

Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures

Environmental cocontamination by polycyclic aromatic hydrocarbons (PAHs) and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by EPA and ATSDR) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), dibenzo[a,h]anthracene (DBahA), benzo[a]anthracene (BaA), and benzo[k]fluoranthene (BkF) has thus been investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84% of 5 microM BaA and 25-40% of 5 microM DBahA was taken up in 24 h. Hepatocytes retained viability up to 1 microM Cd and 5 microM Pb, Hg, or As and 5 microM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 microM PAH concentration was in the order BkF (7.6-fold) > DBahA (6.1 fold) > BaP (5.7 fold) > BbF (3.9-fold) > BaA (2.5-fold). All four metals (1-5 microM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 microM) decreased induction by 47% for BaP, 68% for BaA, 45% for BbF, 79% for BkF, and 53% for DBahA. Induced CYP1A2 protein was much more extensively decreased than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.     

Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.     

Effects of polycyclic aromatic hydrocarbons on liver and lung cytochrome P450s in Mice

Certain polycyclic aromatic hydrocarbons (PAHs) have been reported to induce cytochrome P450 (CYP) 1A1 and 1A2. In the present study, the effects of six well-known PAHs, such as benzo[a]pyrene, benz[a]anthracene, dibenz[a,h]anthracene, chrysene, benzo[k]fluorancene and benzo[b]fluorancene, on the activities of hepatic and pulmonary CYP enzymes were investigated in male ICR mice. When mice were treated intraperitoneally with 3, 10 and 30 mg/kg of individual PAHs for 3 consecutive days, the activities of ethoxyresorufin- and methoxyresorufin-O-dealkylases were significantly and differentially induced in both liver and lung. Moreover, other CYP isozyme-associated monooxygenase activities were also induced significantly in liver and lung with characteristic induction profiles. Our present results suggest that individual PAHs might have inductive effects on CYP isozymes, and that the characteristic inductive effects of individual PAHs on certain CYP isozymes would be developed as a marker for determining exposure to certain PAHs.     

Effects of hydroxylated flavonoids on the ethoxyresorufin O-deethylase and benzo(α)pyrene hydroxylase

In order to understand the mechanism of action of flavonoids on the drug metabolizing enzyme, cytochrome P4501A1, this study was undertaken to examine the effect of chrysin, morin, myricetin and aminopyrine on the activities of ethoxyresorufin O-deethylase and benzo(α) pyrene hydroxylase in the liver. In the isolated perfused rat liver that was pretreated with 3-methylcholanthrene (3MC), chrysin, morin, myricetin and aminopyrine inhibited the activity of ethoxyresorufin O-deethylase with concentration dependent manner. The isolated liver perfusion with chrysin, morin, myricetin and aminopyrine showed inhibition on the induction of ethoxyresorufin O-deethylase by 3MC. And also, in mouse liver hepa I cells, 3MC-stimulated the benzo(α)pyrene hydroxylase activity which was inhibited by chrysin, morin, myricetin and aminopyrine. These results strongly suggested that hydoxylated flavonoids interfered not only the induction of cytochrome P450IA1 enzymes by 3MC but also the interaction of substrates and enzyme.     

Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P), fluoranthene, chrysene and 7,12-dimethylbenz[a]anthracene (DMBA) were acting as antiandrogens in vitro, resulting in IC(50) values of 3.2, 3.9, 4.6, 10.3 and 10.4 microM, respectively. Only at the highest concentration tested (10 microM), a slight inhibitory effect by pyrene, phenanthrene, and anthracene was observed. In contrast, dibenzo[a,h]anthracene (DB[a,h]A) gave rise to an agonistic effect, which was added upon the effect of the androgen receptor agonist R1881 (0.1 nM). The antiandrogenic responses by PAHs (10 microM) were found to be fully reversible, determined in the presence of increasing concentrations of R1881. No cytotoxic effects of the tested compounds were observed as determined either by metabolic reduction using AlamarBlue (up to 20 microM) or determined in cells transfected with a constitutively active hAR (up to 10 microM). The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2,3,7,8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 0.03, 0.4, 0.89, 3.06, and 9.27 microM, respectively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect. In conclusion, no clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (i.e. B [a] A, B [a] P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB [a, h] A), whereas those not able to activate the Ah receptor containing three or four aromatic rings (i.e. pyrene, phenanthrene, anthracene) displayed either very weak or no antiandrogenic effect at concentrations up to 10 microM (with the exception of fluoranthene which blocked the hAR at lower concentrations, but did not activate the Ah receptor).     

Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.     

Inhibition of carcinogen-bioactivating cytochrome P450 1 isoforms by amiloride derivatives

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.     

Cytochrome P450 expression and testosterone metabolism in the liver of deer

Cytochrome P450 expression in cervine liver was investigated using chemical probes and Western blot analysis, and compared with the rat. Deer liver, when compared with rat liver, was characterised by high ethoxyresorufin O-deethylase, coumarin 7-hydroxylase and, to a lesser extent, erythromycin N-demethylase activities; in contrast, deer liver exhibited low debrisoquine 4-hydroxylase, chlorzoxazone 6-hydroxylase and, particularly, lauric acid hydroxylase activities. Ethoxyresorufin O-deethylase activity in deer was markedly inhibited by alpha-naphthoflavone, but was relatively resistant to inhibition by furafylline. Coumarin 7-hydroxylase was inhibited by 8-methoxypsoralen. Western blot analysis using antibodies to rat CYP1A recognised a single, highly expressed protein. Kinetic analysis indicated that a single enzyme is likely to be responsible for the high ethoxyresorufin O-deethylase activity in deer liver. Probing of cervine hepatic microsomes with antibodies to rat CYP2A2 showed that apoprotein levels were higher in the deer compared with the rat. Eadie-Hofstee plot analysis indicated that more than one enzyme catalyses the 7-hydroxylation of coumarin. Western blot analysis using antibodies to rat CYP2B, rat CYP2C11, human CYP2D6, rat CYP3A and rat CYP4A1 revealed in each case the presence of single, poorly expressed, proteins in deer liver. In contrast, when antibodies to rat CYP2E1 were used, a highly expressed single protein was observed. Cervine hepatic microsomes metabolised testosterone to generate androstenedione and a number of hydroxylated products, the major hydroxylation sites being the 2beta-, 6beta- and possibly the 12-position. In summary, this is the first study showing that deer liver expresses all xenobiotic-metabolising cytochrome P450 families, but the level of expression differs from that of the rat.     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.