Cellular localization of cytochrome(s) P-450 metabolizing polycyclic aromatic hydrocarbons in the rat adrenal cortex

Cells were dispersed from the capsular, as well as the inner portion of female rat adrenal glands and subsequently separated on discontinuous Percoll gradients. The adrenal cells were distributed within a density interval ranging from 1.016 to 1.075 g/cm3 and different subpopulations showed distinct morphological appearances in suspension, as well as in culture. The total cells from the inner portion of the adrenals metabolized [14C]7,12-dimethylbenz(a)anthracene at a rate of 4.04 pmol/min 10(6) cells and synthesized corticosterone in response to ACTH stimulation at a rate of 1.07 micrograms/hr/10(6) cells. These activities were 4- and 2.5-fold higher, respectively, than the corresponding activities in cells isolated from the capsular portion. 7,12-Dimethylbenz(a)anthracene monoxygenase activity and ACTH-stimulated steroidogenesis were enriched in two subpopulations of cells obtained on the Percoll gradient and were estimated to be 13.1 pmol/min/10(6) cells and 3.21 micrograms/hr/10(6) cells, respectively, in the most active fraction (at the 1.034/1.040 g/cm3 interface). On the basis of cellular morphology, density and steroidogenic properties, it was concluded that adrenal 7,12-dimethylbenz(a)anthracene monoxygenase activity is localized mainly in the cells of the zona fasciculata.     

Sex-dependent regulation of hepatic cytochrome P-450 by DDT.

Dichlorodiphenyltrichloroethane (DDT) is a well-known inducer of microsomal monooxygenase systems in rodent liver. However, little information is available on its effects on the sex-dependent regulation of CYPs preferentially affected. Therefore, our objective was to evaluate the effects of DDT on the sexual expression pattern of some hepatic P-450 isozymes. Single doses of technical DDT (0, 0.1, 1, 5, 10, or 100 mg/kg body wt) were administered by gavage to Wistar rats. The effects on CYPs 1A1, 2B11/2B2, 2C11, 2E1, 3A1, and 3A2, were assessed 24 h later by means of CYP protein content determined by Western blotting and/or enzyme activities participating in alkoxyresorufin and pnitrophenol metabolism. The highest dose induced 18-fold the expression of CYP3A2 in female rats without producing significant induction (< 3-fold) in males. The effects on this isozyme, which is not normally expressed in females, suggest that DDT is able to modulate sexual metabolic dimorphism, as 3A2 expression is androgen dependent. DDT did not significantly alter CYP3A1 in males, suggesting that DDT is not a pure phenobarbital (PB)-type inducer. The effects on CYP2B1/2B2 protein (19-fold) and associated enzyme activities indicated that males had a lower response threshold than females, but that the latter were able to reach a higher relative induction. The preferential induction of CYPs 2B and 3A by DDT in a sex-related manner suggest that CYP regulation could play an important role in endocrine disruption.     

Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice

P-glycoprotein (PGP), an ATP-dependent membrane transporter is found in epithelial tissues of the liver, kidneys, intestine and blood-brain barrier. In tumor cells, PGP is often overexpressed and confers multidrug resistance toward cancer chemotherapeutics. It has been previously shown in rats that induction of an inflammatory response evokes a decrease in hepatic expression of PGP. In order to identify the inflammatory mediators involved in this phenomenon, we examined the influence of experimentally induced inflammation and pro-inflammatory cytokines (interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha) on the hepatic expression of PGP in mice. A significant reduction in the hepatic expression of mdr1a, mdr1b, mdr2 and spgp genes were seen in endotoxin (lipopolysaccharide (LPS)) and turpentine-treated mice. Similarly, IL-6-treated mice displayed a 70% reduction in protein expression and a 40-70% reduction in the mRNA levels of all PGP mdr isoforms. Administration IL-1beta caused an increase in both mdr1b mRNA and protein expression, however, mRNA levels of mdr1a, mdr2 and spgp were significantly reduced. Administration of TNF-alpha also caused increases in mdr1b mRNA. These findings indicate that IL-6 plays a principal role in the downregulation of PGP that is observed in the livers of mice during an inflammatory response.     

Induction of drug-metabolizing enzymes in Gunn rat liver. Effect of polycyclic aromatic hydrocarbons on cytochrome P-450 regulation

Response of congenitally jaundiced rats (Gunn rats) to administration of polycyclic aromatic hydrocarbons (PAH) was investigated and compared to that of Wistar rats. Unlike Wistar, Gunn males did not exhibit changes in the overall cytochrome P-450 content of hepatic microsomes. The first step in the induction process (i.e. presence of cytosolic receptors for PAH) was found present and functionally similar (number of sites, Kd) to that of Wistar rats from which the Gunn strain is derived. An increase in monooxygenase activities related to P-450c and P-450d isoenzymes specifically induced by PAH was noticed, whereas no effect could be detected on the glucuronidation rate of either 4-nitrophenol, testosterone or estrone. As determined by immunoquantification after Western blotting, the isoenzymatic profile of P-450 from PAH-treated male Gunn rats showed an increase of P-450c and P-450d accompanied by an equivalent decrease in P-4502c (major male-specific isoenzyme). The balance between increase in P-450c and P-450d and decrease in P-4502c may explain the absence of increase in the total P-450 in PAH-treated male Gunn rats. Such a response was not observed in PAH-treated male Wistar rats or in female rats of both strains. In contrast, the response of male Gunn rats to PB treatment was similar to that observed in Wistar rats, i.e. increase in overall cytochrome P-450 content of hepatic microsomes and of specific isoenzyme P-450b/e. A possible regulation of P-450 isoenzyme synthesis by the intracellular haem pool might be involved.     

Polygalasaponin F inhibits secretion of inflammatory cytokines via NF-κB pathway regulation

To study the anti-neuroinflammatory mechanisms of polygalasaponin F (PS-F), ELISA method was used to detect the secretion of inflammatory cytokines. Western blot was used to detect the protein expression and phosphorylation levels. Immunofluorescence assay was used to observe the NF-κB nuclear translocation. PS-F could inhibit the release of inflammatory cytokines TNF-α and NO induced by lipopolysaccharides (LPS) and reduce the expression of inducible nitric oxide synthases (iNOS). As for MAPK-signaling pathway, PS-F could only inhibit the phosphorylation levels of p38 MAPK, but did not significantly affect the phosphorylation levels of JNK and ERK1/2 protein kinases. PS-F could inhibit NF-κB nuclear translocation in a dose-dependent manner. The results of Western blot assay were consistent with immunofluorescence assays. Meanwhile, p38-specific inhibitor SB203580 (20 μM) and p65-specific inhibitor PDTC (100 μM) were, respectively, administered as a positive control. In addition, PS-F could significantly inhibit the cytotoxicity of conditioned medium prepared by LPS-stimulated BV-2 microglia (LPS conditioned media) to neuronal PC12 cells and improve cell viability. PS-F inhibits the secretions of neuroinflammatory cytokines by the regulation of NF-κB-signaling pathway.     

Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons

Nitrated polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that result from various incomplete combustion processes. We have examined the activity of hepatic microsomal enzymes in rats pretreated with a series of environmentally occurring nitrated PAHs including: 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitrofluoranthene, and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of the compounds increased the cytochrome P-450 content more than 2-fold. 1,8-Dinitropyrene, 6-nitrochrysene, and 1- and 3-nitrobenzo[a]pyrene significantly increased arylhydrocarbon hydroxylase activity 2- to 8-fold higher than solvent-treated controls. The induction of 7-ethoxycoumarin O-deethylase activity paralleled that found with arylhydrocarbon hydroxylase. The maximum induction of aminopyrine N-demethylase was only 1.5-fold, and none of the nitrated PAHs caused significant increases in epoxide hydrase or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. The greatest increase was caused by 1-nitrobenzo[a]pyrene followed by 1,3-dinitropyrene, 3-nitrobenzo[a]pyrene and 6-nitrochrysene. These data suggest that nitrated PAHs may potentiate the effects of subsequent exposures to various chemical carcinogens.     

Inhibition of human cytochrome P450 1A1-, 1A2-, and 1B1-mediated activation of procarcinogens to genotoxic metabolites by polycyclic aromatic hydrocarbons

Many chemicals in the environment can cause cancer, and polycyclic aromatic hydrocarbons (PAHs) are among the most ubiquitous. Cancer risk assessments require consideration of these in complex mixtures. PAHs require metabolic activation by cytochrome P450 (P450) enzymes, primarily 1A1, 1A2, and 1B1. We determined if individual PAHs and other procarcinogens affect the activities of human P450s 1A1, 1A2, and 1B1 by measuring 7-ethoxyresorufin O-deethylation (EROD) activity and metabolic activation of PAH dihydrodiols and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) to genotoxic metabolites in a Salmonella typhimurium NM2009 system. Of 23 PAHs examined, benz[a]anthracene (B[a]A), benzo[b]fluoranthene, and 5-methylchrysene were the most potent inhibitors of P450 1A2- and 1B1-catalyzed EROD activity, with IC50 values <10 nM. Other PAHs, e.g., dibenz[a,c]anthracene, dibenz[a,h]anthracene, dibenz[a,j]acridine, and 3-methylcholanthrene, rather selectively inhibited P450 1B1, with IC50 values <15 nM. Benzo[a]pyrene (B[a]P) and nine other PAHs also inhibited P450 1A2 as well as 1B1 with IC50 values <150 nM. Parent PAH compounds were generally more potent than 10 dihydrodiol metabolites of PAHs and 3- and 9-hydroxy B[a]P in inhibiting EROD activity. In addition, we found that three selected PAHs (5-methylchrysene, B[a]P, and B[a]A) inhibited metabolic activation of 5-methylchrysene-1,2-diol, (+/-)-B[a]P-7,8-diol, dibenzo[a,l]pyrene-11,12-diol, and MeIQ to genotoxic metabolites catalyzed by P450s 1A1, 1B1, and 1A2, respectively, in S. typhimurium NM2009. Thus, individual PAHs may affect their own and metabolism of other carcinogens catalyzed by P450 1A1, 1A2, and 1B1, and these phenomena cause alteration in their ability to transform cells when single or complex PAH mixtures are ingested by mammals, influencing risk assessment.     

Modulation of rat hepatic aryl hydrocarbon hydroxylase by various flavones and polycyclic aromatic hydrocarbons

The microsomal cytochrome P-450-dependent aryl hydrocarbon hydroxylase is important in the detoxification of polycyclic hydrocarbons as well as their activation to cytotoxic or carcinogenic derivatives. We have studied compounds that can modify the activity of this enzyme system. Three types of flavones are distinguished on the basis of their effect on the constitutive and polycyclic hydrocarbon-induced rat hepatic enzyme activity: (a) the 5,6- and 7,8-benzoflavones and their more hydrophobic derivatives inhibit the induced enzyme and increase or do not affect the constitutive enzyme activity; (b) derivatives typified by the 4'-hydroxylated benzoflavones similarly decrease both induced and constitutive activities; (c) polyhydroxyflavones inhibit the constitutive enzyme more than the induced enzyme. Two polycyclic hydrocarbons, 9-chloro-7H-dibenzo(a,g)carbazole and 6-aminochrysene, both potent inhibitors of the enzyme system, affect the constitutive and induced enzyme similar to compounds in groups a and b, respectively. The various activity-modulating compounds are useful reagents for distinguishing closely related enzymes present in a variety of different tissues and species under different conditions.     

Modulation of human cytochrome P450 1B1 expression by 2,4,3',5'-tetramethoxystilbene.

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.     

Involvement of cytokines in the hepatic metallothionein expression by alpha-hederin

Alpha-hederin, a triterpenoid saponin, has been reported to induce hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of alpha-hederin on the regulation of MT expression in an in vitro model using the murine hepatoma cell line, Hepa-1c1c7, and the murine macrophage cell line, RAW 264.7. Alpha-hederin that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with alpha-hederin and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in decreased effects of alpha-hederin on MT induction. In the alpha-hederin-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. Accordingly, it was found that the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. These results suggest that the upregulation of MT expression by alpha-hederin is mediated by TNF-alpha and IL-6.     

Mouse renal cytochrome P450IIE1: immunocytochemical localization, sex-related difference and regulation by testosterone

Cytochrome P450IIE1 is responsible for the metabolic activation of N-nitrosodimethylamine and a variety of other chemicals. Renal P450IIE1 was shown previously to be regulated by testosterone in C3H/HeJ and BALB/c mice. The present study investigated the distribution of cytochrome P450IIE1 in the kidneys of C3H/HeJ and BALB/c mice. The amount of P450IIE1 was immunotitrated by immunohistochemistry using polyclonal antibodies against rat P450IIE1. Strong immunoreactivity was identified mainly in the cortical tubules, including proximal tubules and some tubules. Weak immunoreactivity was also observed in the outer medulla when higher concentrations of antibodies were used. Much higher immunostaining was observed in male mice than in female mice when identical antibody dilutions were used. The renal P450IIE1 level in females was elevated to the same level as that in males 24 hr after administration of testosterone. The results showed a specific cellular localization of cytochrome P450IIE1 in mouse kidney. The findings may lead to a better understanding of the site-specific renal toxicity and carcinogenesis due to the activation of chemicals by cytochrome P450IIE1.     

Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 in the guinea pig.

Addition of simple aromatic hydrocarbons (benzene, ethylbenzene, naphthalene) to guinea pig adrenal microsomes produced typical Type I difference spectra (ΔOD_385-420). Spectral dissociation constants (K_s) for each indicated a far higher affinity for adrenal than hepatic cytochrome P-450. Hydrocarbon affinities for adrenal cytochrome P-450 were similar to that for progesterone, an endogenous steroid substrate. Ethylmorphine and aniline produced Type I and Type II spectral changes respectively in adrenal microsomes. The K_s, and magnitude of spectrum for each in adrenals was similar to that in livers. Nonetheless, demethylation of ethylmorphine proceeded far more rapidly in adrenal than hepatic tissue. The Michaelis constants (K_m) for ethylmorphine metabolism in both tissues were similar. Although the aniline-induced difference spectra in adrenal and hepatic microsomes did not differ substantially, aniline hydroxylase activity was far greater in liver. Pretreatment of guinea pigs with phenobarbital or 3-methylcholanthrene increased hepatic but not adrenal ethylmorphine metabolism. Spironolactone pretreatment, in contrast, did not affect hepatic metabolism, but significantly lowered adrenal demethylase activity. The results indicate a relative non-specificity of guinea pig adrenal microsomal cytochrome P-450 and suggest that the adrenal cortex may represent a significant site for the extra-hepatic metabolism of foreign compounds in the guinea pig.     

Involvement of cytokines in the hepatic expression of metallothionein by ursolic acid

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have inducing activity of hepatic metallothionein (MT) which responsible for the detoxification of heavy metals; however, the mechanism underlying its effects is poorly understood. To further determine the underlying mechanism of UA, this study investigated the effects of UA on the induction of hepatic MT expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. The UA added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels were markedly increased when Hepa-1c1c7 cells were cultured with UA-treated conditioned media from RAW 264.7. Concomitant treatment with UA and pentoxifylline, a TNF-alpha synthesis inhibitor, to RAW 264.7 cells decreased the effects of UA on the MT induction. In UA-exposed RAW 264.7 cell cultures, TNF-alpha and IL-6 production and TNF-alpha and IL-6 mRNA levels increased. When antibodies to TNF-alpha or/and IL-6 were added to UA-treated conditioned media from RAW 264.7, the MT induction activity was inhibited. These results demonstrate that UA induces hepatic MT expression through TNF-alpha and IL-6 released from UA-activated macrophages, which may be the mechanism, whereby UA elicits its biological effects.     

Modulation of cytochrome P450 1A1 by food-derived heterocyclic aromatic amines

A short-term effect of a meal of fried meat is a postprandial induction of hepatic and intestinal cytochrome P450 activity. In order to identify the components responsible for this effect we investigated the potency of food derived genotoxic heterocyclic aromatic amines (HA) to induce CYP1A1 in vitro. In two cell lines, the rat hepatoma cell line H4IIE and the human breast cancer cell line MCF-7, we investigated 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and Harman representing the different classes of HA at concentrations from 10(-8) to 10(-4) M. Induction of CYP1A1 was analysed on the mRNA level by semi-quantitative RT-PCR and the protein level (western blot using specific antibodies). The relative order of enzyme induction was Trp-P-1 with 1.4 x 10(-6) M (EC50 compared to TCDD 10(-9) M), MeAalphaC (1.4 x 10(-5)), Harman (2.1 x 10(-4)) and MeIQx (1.0 x 10(-3)). Furthermore, CYP1A1 enzyme activity was analysed as ethoxyresorufin-O-deethylase. While protein and mRNA analyses gave similar results, competitive inhibition impaired the enzyme activity assay. Inhibition of CYP1A1 activity was determined using microsomes of heterologous expressed CYP1A1. This dose-dependent inhibitory activity paralleled the induction potency. These results compare well with earlier data published for hepatic enzyme induction by HA observed in animal experiments. However, since the observed activities are rather weak and the amounts of HA ingested with a meal are low, there may be other factors involved in the observed postprandial enzyme induction in humans. On the other hand, concentrations in the micromolar range that are reached in high dosage animal experiments with HA may well influence cytochrome activity and, thus, influence the experimental outcome of these studies.     

Gemfibrozil and its glucuronide inhibit the hepatic uptake of pravastatin mediated by OATP1B1

When pravastatin (40?mg/day) was co-administered with gemfibrozil (600?mg, b.i.d., 3 days) to man, the AUC of pravastatin increased approximately 2-fold. We have clarified that OATP1B1 is a key determinant of the hepatic uptake of pravastatin in humans. Thus, we hypothesized that gemfibrozil and the main plasma metabolites, a glucuronide (gem-glu) and a carboxylic acid metabolite (gem-M3), might inhibit the hepatic uptake of pravastatin and lead to the elevation of the plasma concentration of pravastatin. Gemfibrozil and gem-glu inhibited the uptake of ¹⁴C-pravastatin by human hepatocytes with K_i values of 31.7?µM and 15.7?µM, respectively and also inhibited pravastatin uptake by OATP1B1-expressing Xenopus laevis oocytes with K_i values of 15.1?µM and 7.6?µM. Additionally, we examined the biliary transport of pravastatin and demonstrated that pravastatin was transported by MRP2 using both human canalicular membrane vesicles (hCMVs) and human MRP2-expressing vesicles. However, gemfibrozil, gem-glu and gem-M3 did not affect the biliary transport of pravastatin by MRP2. Considering the plasma concentrations of gemfibrozil and gem-glu in humans, the inhibition of OATP1B1-mediated hepatic uptake of pravastatin by gem-glu would contribute, at least in part, to the elevation of plasma concentration of pravastatin by the concomitant use of gemfibrozil.