Monooxygenase and UDP-glucuronyltransferase activities in established cell culture

Cells in culture were investigated for the expression of monooxygenase and UDP-glucuronyltransferase activities as representatives of activating and inactivating pathways of drug metabolism. Most established cell lines express monooxygenase activity that is detected by the oxygenation of polycyclic hydrocarbons and appears to be a function of cytochrome P-448-dependent enzyme forms (Wiebel et al., 1977). In the hepatoma cell line, H-4-II-3, dexamethasone is capable of increasing the level of benzo(a)pyrene-monooxygenation about 10-fold and of potentiating its induction by benzo(a)anthracene. The enzyme activities elicited by dexamethasone and the polycyclic hydrocarbon did not significantly differ in their response to 7,8-benzoflavone, an inhibitor of cytochrome P-448-dependent monooxygenases. Observations on the pattern of benzo(a)pyrene metabolites formed in benz(a)anthracene-treated H-4-II-E and BHK21(C13) cells indicate that established cell cultures may contain different forms of monooxygenases of the cytochrome P-448 type. The majority of cell lines tested express UDP-glucuronyltransferase activity directed toward the substrate, 3-hydroxybenzo(a)pyrene. No clear correlation appears to exist between the presence and level of monooxygenase and glucuronyltransferase activities in the various cell lines, i.e., the cultures may express any one or both enzymes. The ratio of the two enzyme levels can be modified by selective induction. Thus, at present there is a choice of established cells in culture exhibiting widely differing ratios of activating and inactivating enzymes to analyse the metabolic pathways of selected classes of foreign compounds, such as polycyclic hydrocarbons, and to determine their toxicological significance. Further efforts are likely to yield cell lines that express the enzymic functions lacking in the cultures currently used and will be suitable to study the full spectrum of foreign compounds.     

Aryl hydrocarbon (benzo[a]pyrene) hydroxylases in liver from rats of different age, sex and nutritional status. Distinction of two types by 7,8-benzoflavone.

Two types of aryl hydrocarbon (benzo[a]pyrene) hydroxylase have been distinguished in liver from rats of different sex and age by their sensitivity to the synthetic flavonoid, 7,8-benzoflavone. One type, which is stimulated by the 7,8-benzoflavone, is found in newborn rats and predominates in the liver of adult male rats. This type is inducible by phenobarbital. A second type, which is inhibited by 7,8-benzoflavone, comprises a larger fraction in the liver of adult female rats and is inducible by polycyclic hydrocarbons in immature and mature animals of either sex. The presence of this form in adult female liver is also indicated by the kinetics of the hydroxylase reaction. Removal of solid food for 18 hr not only decreases hepatic aryl hydrocarbon hydroxylase activity in female rats, but also lowers the degree of inhibition by 7,8-benzoflavone. Kinetic data suggest that at low concentrations 7,8-benzoflavone acts as a competitive inhibitor but at higher concentrations inhibits the hydroxylation reaction by a more complex mechanism.     

Maternal cigarette smoking,placental aryl hydrocarbon hydroxylase and neonatal size

Aryl hydrocarbon hydroxylase (AHH) activity was determined in placental samples from smokers and non-smokers. The offspring from pregnancies in which placental AHH was elevated weighed about 400 g less and were over 1 cm shorter than those from non-smoking mothers, whereas those from AHH-negative pregnancies were of the same size as those from non-smoking mothers. It is suggested that placental AHH be used as a measure of foetal exposure to maternal cigarette smoking.     

Genetic differences between C57BL/6 and DBA/2 mice in the inductions of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene,p-nitrophenol,and bilirubin by 3-methylcholanthrene

In C57BL/6 mice, aryl hydrocarbon hydroxylase (AHH) increased 1 day after treatment with 3-methylcholanthrene, and induction of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene, p-nitrophenol and bilirubin in the liver microsomes was observed 2 to 5 days later. In DBA/2 mice, neither AHH nor transferase activities were influenced by 3-methylcholanthrene. These results suggest that induction of activating and detoxicating enzymes is genetically linked.     

Effect of maternal cigarette smoke exposure on foetuses of inbred strains of mice responsive and non-responsive to polycyclic aromatic hydrocarbons

To evaluate the role of genetically controlled responsiveness to polycyclic aromatic hydrocarbons (PAH) in the effect of cigarette smoke on birth weight, pregnant mice were exposed to cigarette smoke from day 1 to day 17 of pregnancy and the offspring studied on day 18. An unequivocal phenotyping of foetuses with respect to responsiveness was achieved by measuring the activity of aryl hydrocarbon hydroxylase (AHH) in the foetal liver and corresponding placenta after pretreatment of the dam with β-naphthoflavone. The foetuses of dams exposed to standard non-filter cigarettes were consistently smaller than those of the controls, whereas smoke from commercial filter cigarettes was only marginally effective. No statistically significant association with the responsiveness to PAH could be found with respect to this effect on weight. These results suggest that genetically determined responsiveness to PAH, although of importance for PAH foetotoxicity in mice, is not important in the effect of cigarette smoke on birth weight in the strains studied.     

Aryl hydrocarbon receptor-independent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) constitute a major family of widely‐distributed environmental toxic contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). B(a)P has been recently shown to trigger an early and transient increase of intracellular calcium concentration ([Ca²⁺]_i), involved in AhR‐related up‐regulation of target genes by B(a)P. This study was designed to determine whether AhR may play a role in [Ca²⁺]_i induction provoked by B(a)P. We demonstrated that, in addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up‐regulated [Ca²⁺]_i in human endothelial HMEC‐1 cells. Moreover, α‐naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca²⁺]_i. Knocking‐down AhR expression in HMEC‐1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P‐mediated induction of [Ca²⁺]_i, whereas it efficiently counteracted B(a)P‐mediated induction of the referent AhR target gene cytochrome P‐450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca²⁺]_i induction in an AhR‐independent manner. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

The effect of dietary protein and fat on the activity of aryl hydrocarbon hydroxylase in rat liver,kidney and lung

Feeding low protein diets reduces the activity of aryl hydrocarbon hydroxylase (3,4 benzo(α)pyrene hydroxylase) in rat liver and lung, but not in the kidney. In the kidney the level of aryl hydrocarbon hydroxylase activity is increased by feeding a diet containing fats. This increase in the kidney aryl hydrocarbon hydroxylase activity occurs when the fat content of the diet rises above 4 per cent and is maximal after feeding such diets for 7 days.     

Induction of CYP1A1 and CYP1B1 in liver and lung by benzo(a)pyrene and 7,12-d imethylbenz(a)anthracene do not affect distribution of polycyclic hydrocarbons to target tissue: role of AhR and CYP1B1 in bone marrow cytotoxicity

Polycyclic aromatic hydrocarbons (PAHs) (50 mg/kg, i.p.) selectively deplete mouse bone marrow (BM) hematopoietic cells through a process that is dependent on CYP1B1. 7,12-dimethylbenz(a)anthracene (DMBA), which forms greater amounts of dihydrodiol-epoxide-DNA adducts in BM, is much more effective in depleting BM cells than benzo(a)pyrene (BP). BM toxicity by BP is restored in congenic mice expressing a weakly responsive aryl hydrocarbon receptor (AhR(d) replaces AhR(b)). BP strongly induces CYP1A1 around the hepatic vein whereas DMBA produces a weaker diffuse response, paralleling differences in CYP1A1 protein. These responses are absent in AhR(d) mice. BP and DMBA broadly and equally induce CYP1A1 in the lung, while CYP1B1 is induced in bronchial blood vessels. In sternum, CYP1B1 is induced in BM and white fat, whereas CYP1A1 is induced only in brown fat. BP and DMBA levels were similar within blood, lung, and BM and did not rise in AhR(d) mice. In liver, selective decrease of BP was consistent with induced metabolism via CYP1A1, which nevertheless does not determine the blood levels and distribution to BM. Effective delivery of BP to BM is indicated by formation of BP-quinone DNA adducts and the effective induction of CYP1B1. The low formation of BP-dihydrodiol-epoxide-DNA adducts suggests effective AhR induction of BM detoxifying reactions that prevents their formation from dihydrodiols. These findings contrast with the substantial hepatic CYP1A1 contribution for PAHs previously seen for intragastric administration where first pass elimination limits the amount of PAHs reaching the BM.     

Effects of vitamin A deficiency on hepatic and extrahepatic mixed-function oxidase and epoxide-metabolizing enzymes in guinea pig and rabbit.

Male guinea pigs and male rabbits were fed a vitamin A deficient diet for 9 weeks and for 12 weeks respectively. Hepatic levels of vitamin A were significantly reduced in the vitamin A deficient animals. The activities of some xenobiotic-metabolizing enzymes were measured in the liver, lung and small intestine. Aryl hydrocarbon hydroxylase, aniline hydroxylase, and 7-ethoxycoumarin deethylase activities were decreased in the vitamin A deficient guinea pig liver. However, in the guinea pig small intestine, aniline hydroxylase, 7-ethoxycoumarin deethylase, aminopyrine demethylase, and aryl hydrocarbon hydroxylase specific activities were increased. In rabbits, vitamin A deficiency decreased hepatic aniline hydroxylase and 7-ethoxycoumarin deethylase activities but increased intestinal aminopyrine demethylase activity. Enzyme activities in lung were not altered by vitamin A deficiency in guinea pig or rabbit. Microsomal epoxide hydrase and microsomal supernatant glutathione S-transferase activities in the three tissues of both species were not altered by vitamin A deficiency.     

Aryl hydrocarbon hydroxylase induction in mammalian liver-derived cell cultures. Effects of various metabolic inhibitors on the enzyme activity in hepatoma cells.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction in one mouse hepatoma and two rat hepatoma cell lines was characterized with respect to optimal growth requirements and optimal inducing concentrations of polycyclic hydrocarbons, phenobarbital, biogenic amines, and numerous other hydrophobic compounds. Cordycepin, ethidium bromide and puromycin aminonucleoside treatment of the mouse tumor line Hepa-1 produce strikingly different effects on the kinetics of hydroxylase induction, when the inducers benz[a]anthracene, phenobarbital and iso-proterenol are compared. without any of these ‘usual inducers’ present in the mouse hepatoma cell line, 0.50 to 5.0 mM puromycin aminonucleoside or 40 nM actinomycin-D causes large increases in the hydroxylase activity (at a time when gross RNA synthesis is virtually 100 per cent inhibited and gross protein synthesis is more than 50 per cent inhibited). This aminonucleoside- or actinomycin-D-mediated induction process induction, when the inducers benz[a]anthracene, phenobarbital and isoproterenol are compared. With-without prior treatment with the usual inducers and hence presumably without prior accumulation of putative induction-specific RNA: moreover, this induction process apparently requires little, or no. simultaneous RNA synthesis during exposure to the aminonucleoside or actinomycin-D. In contrast, when primary cultures derived from normal rat liver are treated with similar high concentrations of the aminonucleoside, no induction of the hydroxylase activity occurs. This result suggests that the effect of puromycin aminonucleoside on hydroxylase-specific mRNA synthesis, processing or stabilization is very different between the hepatoma cell line and normal fetal rat primary hepatocytes in culture.     

Interleukin-8 induction by the environmental contaminant benzo(a)pyrene is aryl hydrocarbon receptor-dependent and leads to lung inflammation

Benzo(a)pyrene (BP) is an environmental contaminant known to favor airway inflammation likely through up-regulation of pro-inflammatory cytokines. The present study was designed to characterize its effects toward interleukin-8 (IL-8), a well-established pulmonary inflammatory cytokine. In primary human macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion levels in a dose-dependent manner. Such an up-regulation was likely linked to aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was reduced after AhR expression knock-down through RNA interference. Moreover, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation experiments showed BP-triggered binding of AhR to a consensus xenobiotic responsive element (XRE) found in the human IL-8 promoter. Finally, BP administration to mice led to over-expression of keratinocyte chemoattractant (KC), the murine functional homologue of IL-8, in lung. It also triggered the recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was however fully abolished in the presence of a chemical antagonist of the KC/IL-8 receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of KC in BP-induced lung inflammation. Overall, these data highlight an AhR-dependent regulation of IL-8 in response to BP that likely contributes to the airway inflammatory effects of this environmental chemical.     

Influence of estradiol on the benzo(a)pyrene hydroxylase activity induced by hexachlorocyclohexane

Basal BP-hydroxylase activity was measured in male Swiss mice from the age of 3 weeks to 20 months. Maximal enzyme activity was at the age of 5 months. Comparison of the inducibility of BP-hydroxylase by HCH was also investigated in male and female mice of different ages. Male mice showed higher induction of BP-hydroxylase by HCH than females of the same ages. Sterilization of female mice enhanced enzyme induction. Estradiol exhibited competitive inhibition of BP-hydroxylase activity. After treatment with HCH for 8 months, female mice had a lower tumour incidence than males, and this paralleled a lower induction of BP-hydroxylase.     

Prenatal induction of benzo(a)pyrene hydroxylases in mice

1. Benzo(a)pyrene hydroxylase (BPH) activity was measured in homogenates of fetal liver (day 18) or of whole-embryos of mice on day 9, 10 or 12 of gestation after maternal pretreatment with B(a)P on 3 consecutive days. A³H-liberation assay with³H-B(a)P labelled either generally or at the 6-position was used. The values obtained with the embryonic/fetal tissues were compared with those found in maternal liver. 2. Three oral doses of 17.5 mg B(a)P/kg body wt were found to just significantly induce BPH in maternal liver. An induction was observed after pretreatment with 24 mg B(a)P/kg body wt in 9-, 10-or 12-day-old whole-embryos, but the V_max reached was only 10–20% (1% on day 9) of that of adult non-induced liver. The K_m (6-hydroxylation) for all tissues tested were in the same range (600–900 nM). The induction was demonstrable in embryos at tissue levels about one order of magnitude lower than those required for induction in maternal liver. 3. Treatment with 25 mg B(a)P/kg body wt on 3 consecutive days was required to induce BPH in fetal liver on day 18 of gestation. The required B(a)P tissue concentrations were about one half of those necessary for induction in maternal liver. 4. Among a variety of other polycyclic hydrocarbons only chrysene showed an inducing potency similar to that of B(a)P in adult and fetal liver. For all compounds tested there was no correlation found in the inducing potency between adult and fetal liver (e.g. coronene). 5. The doses required to induce BPH in the maternal or fetal liver or in whole embryos of rodents are significantly higher (mg range) than those of usual average human exposure or those taken up by smokers (ng range).Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo(a)pyrene - BPH benzo(a)pyrene hydroxylases - PAH polycyclic aromatic hydrocarbons     

Inhibition of human mesenchymal stem cell-derived adipogenesis by the environmental contaminant benzo(a)pyrene

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are environmental contaminants exerting various toxic effects. PAHs have notably been found to inhibit adipogenesis in rodent species. To determine whether a similar process concerns human cells, we have analyzed the effects of BP towards differentiation of human cultured mesenchymal stem cells (MSC) into adipocytes, triggered by a pro-adipogenic culture medium. BP was found to markedly prevent formation of lipid vesicles, cellular lipid accumulation and up-regulation of adipogenic markers such as fatty acid binding protein-4 and glyceraldehyde-3-phosphate dehydrogenase, which represent major hallmarks of human MSC-derived adipocytes. The aryl hydrocarbon receptor (AhR), known to mediate most of the toxic effects of PAHs, was demonstrated to be present and functional in human MSC. 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR agonist like BP, was found to inhibit lipid accumulation in human MSC cultured with adipogenic medium, in contrast to the PAH benzo(e)pyrene, known to not, or only poorly, interact with AhR. Moreover, BP inhibitory effect toward lipid accumulation in MSC exposed to adipogenic medium was fully counteracted by co-treatment with the AhR antagonist α-naphtoflavone. Taken together, these data indicate that environmental PAHs like BP can likely inhibit human adipogenesis in an AhR-dependent manner.     

Influence of pretreatment with tobacco smoke condensate or 3-methylcholanthrene on [14C] benzo (A) pyrene metabolism in the isolated perfused rabbit lung

Pretreatment 24 h before sacrifice with i.p. tobacco smoke condensate (TSC) or 3-methylcholanthrene (3MC) increased the rate of disappearance of [¹⁴C]benzo(a)pyrene (BP) from an isolated perfused rabbit lung model. Both pretreatments significantly increased the amount of most metabolites formed. This study indicates that rabbits tend to resemble rats, mice and hamsters in that increased rates of pulmonary BP metabolism are a consequence of exposure to TSC.     

Increased aryl hydrocarbon hydroxylase and prolyl hydroxylase activities in lung organ cultures exposed to benzo[a]pyrene

Exposure of neo-natal rat lungs in organ culture to 10–25 μM benzo[a]-pyrene (BaP) elevated the activities of aryl hydrocarbon hydroxylase (AHH) and prolyl hydroxylase (PH). Pyrene, a non-carcinogenic hydrocarbon did not elicit this response. Prolyl hydroxylase is an indicator of collagen synthesis and increased PH activity in the lungs reflects increased collagen synthesis. Our studies suggest that the earliest events in BaP-induced lung injury may include altered collagen metabolism.     

Induction of cytochrome P-450 related metabolic activities in the rat ventral prostate

Rats were treated with β-naphthoflavone (BNF) or phenobarbital (PB), $\text{80}\text{mg}\text{kg}$ i.p. for 4 days and microsomes prepared from the ventral prostate were assayed for aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin-O-deethylase (7-EOD) and NADPH-cytochrome c reductase activities. The relative increase after BNF treatment was approx. 1000 times for AHH, and 800 times for 7-EOD, while the NADPH-cytochrome c reductase activity was not significantly altered. PB treatment caused no significant effects. Treatment with BNF led to an increased in vitro formation of all measured benzo(a)pyrene (B(a)P) metabolites, especially phenols. Carbon monoxide (CO) and α-naphthoflavone (α-NF) inhibited 7-EOD- and AHH-activities. The rat ventral prostate contains inducible cytochrome P-450-dependent enzymes, a circumstance of potential importance in the etiology of prostatic carcinoma.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes)

When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105000g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 g/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non-toxic congener 1,3,6,8-TCDD at concentrations as high as 50 g/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putativeAh receptor in these fish embryos. At 50 g/l, -naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8-TCDD did at toxic concentrations. However, at 50 g/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.Presented in part at the 28th Annual Meeting of the Society of Toxicology, Atlanta, GA, 1989.