液化石油气燃烧颗粒提取物对大鼠肝,肺药物代谢酶的诱导

在5～40mg/kg剂量范围内,液化石油气(LPG)燃烧颗粒物的提取物对大鼠肺 AHH和GST具有明显的诱导作用,其中对肺AHH的诱导大大超过对GST的诱导。当总染毒剂量相同时,少量多次诱导的酶活性高于一次大剂量诱导的酶活性。各剂量组肝AHH和GST活性与对照组相比未见明显变化。结果提示,LPG燃烧颗粒物进入肺脏后可诱导并改变其有关代谢酶的代谢模式,从而加速PAH和NO_2-PAH等致癌物质在肺内的代谢活化。     

Effects of chlorinated paraffins on liver weight,cytochrome P-450 concentration and microsomal enzyme activities in chick embryos

Sublethal doses of three technical preparations of chlorinated paraffins (CPs) (Cereclor 42 (C_22–26, 42% Cl w/w), Cereclor 50LV (C_10–13, 49% Cl w/w) and Cereclor 70L (C_10–13, 70% Cl w/w)) were injected into the yolks of hens' eggs after 4 days of incubation. The liver weight, the cytochrome P-450 concentration in the liver and the liver microsomal activity of aminopyrine N-demethylase (APND), aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) were determined in chick embryos incubated for 20 days. The degree of chlorination and probably also the carbon chain length of the CPs were of importance for their effects. Cereclor 70L was the most potent in causing increases in liver weight, cytochrome P-450 concentration and APND activity. Cereclor 42 was the least potent in these respects, even causing reduced APND activity. A decrease in AHH activity occurred in chick embryos treated with Cereclor 50LV, and a reduction in ECOD activity was noted as a result of treatment with Cereclor 42 and Cereclor 50LV.     

A comparison of the effects of hexachlorobenzene, beta-naphthoflavone, and phenobarbital on cytochrome P-450 and mixed-function oxidases in Japanese quail

Hexachlorobenzene (HCB), beta-naphthoflavone (BNF), or phenobarbital (PB) was administered to Japanese quail to determine their effects on hepatic porphyrin levels and drug-metabolizing enzymes. While HCB increased porphyrin levels, PB slightly reduced them, and BNF had no effect. HCB was an excellent inducer in quail, increasing the specific content of cytochrome P-450 to levels similar to those produced by BNF. Additional similarities between HCB- and BNF-treated quail included a comparable hypsochromic absorption shift in the CO-reduced difference spectra of cytochrome P-450 and similar effects on the activities of cytosolic glutathione S-transferase (GSH-t), biphenyl hydroxylase (BPH), and ethoxyresorufin O-deethylase (EROD). However, a differential response to HCB and BNF treatment was seen in the activities of hepatic NADPH-cytochrome P-450 reductase, epoxide hydrolase, GSH-t (microsomal), aryl hydrocarbon hydroxylase (AHH), and ethoxycoumarin O-deethylase (ECOD). The activities of NADPH-cytochrome P-450 reductase, AHH, and ECOD following treatment with HCB were similar to those found after dosing with PB. HCB caused a pattern of induction that was distinct from either BNF or PB and appeared to be a "mixed-type" inducer. The rapidity of the HCB-induced porphyrogenic response of Japanese quail, as compared to mammals, may provide unique advantages for making correlations between the in vivo metabolism of haloaromatic hydrocarbons and their effects on porphyrin metabolism.     

Polychlorinated aromatic hydrocarbon lethality, mixed-function oxidase induction, and uroporphyrinogen decarboxylase inhibition in the chick embryo: dissociation of dose-response relationships

Effects on survival of chick embryos and on the activity of hepatic enzymes involved in heme biosynthesis and hemoprotein function were compared as a function of dose of 3,4,3',4'-tetrachlorobiphenyl (TCB), 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 2,3,6,2',3',6'-HCB, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 24 hr and at 9 days after polyhalogenated aromatic hydrocarbon (PAH) administration. 2,3,6,2',3',6'-HCB did not alter enzyme activities or survival. The other PAH increased delta-aminolevulinic acid synthetase up to 10- to 20-fold after 24 hr or 9 days of exposure. Hepatic porphyrins and uroporphyrinogen decarboxylase (Uro-D) activity were unaffected except by 3,4,3',4'-TCB which after 9 days of exposure increased porphyrins slightly (less than 2-fold) at 500 and 1000 nmol/egg and decreased Uro-D by 20% at 1000 nmol/egg. 3,4,3',4'-TCB, 3,4,5,3',4',5'-HCB, and TCDD preferentially induced cytochrome P-448-mediated mixed-function oxidases. The degree of induction was the same or greater after 9 days of exposure than after 24 hr. The three PAH increased aminopyrine demethylase and 7-ethoxycoumarin deethylase up to 2-fold and aryl hydrocarbon hydroxylase (AHH) 10- to 12-fold. 7-Ethoxyresorufin deethylase (7-ER) was increased 45-fold by 3,4,3',4'-TCB, 28-fold by 3,4,5,3',4',5'-HCB, and 55-fold by TCDD. The three PAH decreased survival after 9 days but not after 24 hr. Decreases in survival were accompanied by decreased thymus weights and increased incidences of pericardial and subcutaneous edema in surviving embryos. 3,4,3',4'-TCB caused dose-related decreases in survival at 100 to 1000 nmol/egg. 3,4,5,3',4',5'-HCB decreased survival at 500 and 1000 nmol/egg and TCDD at 6 nmol/egg. The effects on survival were greatest for 3,4,3',4',-TCB and least for TCDD, notwithstanding that all three PAH induced AHH to the same degree and that TCDD caused the greatest induction of 7-ER. The results demonstrate that the dose-response relationships for hepatic induction and lethality are dissociated and that the maximal induction levels are not correlated with the incidence of lethality. The findings indicate that the induction per se does not lead directly to toxicity and that other factors must intervene. The results further show that PAH lethality occurs independently of effects on hepatic Uro-D.     

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.     

Activity of aldrinepoxidase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase during the development of chick embryos in ovo

Since the chick embryoin ovo is susceptible to the action of some agents needing metabolic activation we studied the development of the activity of cytochrome P450-dependent monooxygenases in embryo/fetal tissue. The activities of aldrinepoxidase (AE), 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) were measured in whole embryos, liver and yolk sac tissue of the chick embryo during developmentin ovo from day 4 to day 15 of incubation (DI). In yolk sac tissue enzyme activities could be detected from DI 4 on. While EROD activity was only marginally developed, AE and ECOD activities were more pronounced in the earlier developmental period and showed a clear decrease by the time the liver activities rose. With the methods used AE activity could be measured in the homogenate of the whole embryo proper from DI 4 on while EROD and ECOD activity was not detectable before DI 6 or DI 7, respectively. In liver tissue enzyme activities of the three monooxygenases studied developed to a considerable degree from DI 9 on and tended to exhibit maximum values around DI 11–13. Studies on monooxygenase activities in extra-embryonically located tissues have not been published until now. The importance of the yolk sac as a metabolically relevant organ during embryogenesis is pointed out in this study.     

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using ³²P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had ³²P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.     

The interaction of L-triiodothyronine and 2,3,7,8-tetrachlorodibenzo-p-dioxin on Ah-receptor-mediated hepatic Phase I and Phase II enzymes and iodothyronine 5'-deiodinase in thyroidectomized rats.

Across all levels of L-triiodothyronine (L-T3) treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased hepatic cytochrome P-450-associated activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-dealkylase (EROD) and aryl hydrocarbon hydroxylase (AHH). The treatment of thyroidectomized rats with L-T3 at physiologic replacement levels in concert with TCDD produced an increase in ECOD, EROD and AHH activity above that seen with only TCDD. TCDD as well as L-T3 enhanced the activity of hepatic 1-naphthol glucuronyl transferase (NGT). In addition, the combined effect of L-T3 and TCDD resulted in similar levels of induction of NGT at both physiologic and supraphysiologic doses of L-T3. TCDD treatment resulted in elevated serum T3 levels at both physiologic and supraphysiologic levels of L-T3. One TCDD dose inhibited hepatic microsomal 3,3',5'-triiodothyronine (reverse T3) 5'-deiodinase activity by 61% in thyroidectomized, T3-untreated rats. The inhibition of 5'-deiodinase activity was partially overcome by increasing the T3 dose.     

Induction effects of polychlorinated biphenyls,polycyclic aromatic hydrocarbons and other widespread aromatic environmental pollutants on microsomal monooxygenase activities in chick embryo liver

 Cytochrome P450-dependent 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-dealkylase (PROD) and 7-ethoxycoumarin O-deethylase (ECOD) activities in 14-day-old chick embryo livers were determined 24 h after pretreatment with selected widespread aromatic environmental contaminants, including polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), hexachlorobenzene, and dialkylesters of phthalic acid, and compared with the inducing potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar and mono-o-chlorinated PCBs. The effects of other model inducers, i.e. phenobarbital and pyrazole, were also examined. Specificity of EROD induction was estimated with regard to contaminants frequently present in environmental samples and dose-response curves for EROD induction were determined. A strong induction (comparable with that by mono-o-chlorinated biphenyl treatment) by dibenzo[a,h]anthracene, benzo[k]fluoranthene or benzo[b]fluoranthene was found, but the maximal level of EROD activity inducible by TCDD was not achieved, partly due to the high toxicity of the tested PAHs. 3-Methylcholanthrene showed moderate inducing potencies; benz[a]anthracene, benzo[a]pyrene, chrysene and 2,2′,3,4,4′,5′-hexachlorobiphenyl appeared to be weak inducers. Other PAHs and PCBs tested, as well as hexachlorobenzene, dialkyl phthalates, phenobarbital and pyrazole had no marked effects on the EROD level. ECOD activities were increased non-specifically by TCDD, 3-methylcholanthrene, hexachlorobenzene and phenobarbital. A significant enhancement of PROD activity by TCDD and related inducers was observed, while phenobarbital induced the PROD activity only weakly; SDS-PAGE analysis showed that the chicken phenobarbital-inducible cytochromes P4502H with apparent molecular weights 50 kDa were not markedly induced by the TCDD- or 3-methylcholanthrene treatments. Inhibition of EROD and PROD by 9-hydroxyellipticine, a specific inhibitor of rat hepatic cytochrome P4501A1, revealed that PROD induction by TCDD and other P4501A-inducers was probably a result of a broader substrate specificity of chick embryo P4501A. Measurement of EROD activities in chick embryo liver is highly sensitive, specific and suitable for the determination of TCDD-type toxicity of new drugs, agrochemicals, and industrial pollutants. Received: 4 January 1995 / Accepted: 28 September 1995     

沈阳市大气悬浮颗粒物中硝基多环芳烃的观察

采用ＴＡ９８及其缺乏硝基还原酶的衍生菌株ＴＡ９８ＮＲ和ＴＡ９８／１，８ＤＮＰ６，对沈阳市不同功能分区１９９０年第一季度大气悬浮颗粒物进行致突变研究。结果显示，颗粒物的二氯甲烷提取物对上述三菌株均具有明显直接致突变作用。其中对ＴＡ９８ＮＲ和ＴＡ９８／１，８ＤＮＰ６的直接回变数比其母株均降低，ＴＡ９８／１，８ＤＮＰ６的直接回变数降低更明显。提示提取物中含有直接致突变的硝基多环芳烃，其中硝基芘类特别是二硝基芘类在提取物的致突变活性中占重要地位。结果还表明二硝基芘类和１－硝基芘类污染以商业交通区最严重，其次为居民区、工业区。风景区污染相对明显减少。     

Pulmonary aryl hydrocarbon hydroxylase activity of mice, rats and guinea pigs following long term exposure to mainstream and sidestream cigarette smoke

Male, C57BL mice, Sprague-Dawley rats and Hartley guinea pigs were given daily exposure to either mainstream or sidestream whole smoke from University of Kentucky Reference cigarette, 2R1, for 16 weeks. Pulmonary aryl hydrocarbon hydroxylase (AHH) activity was assayed in lung tissue 9000 g supernatants by measuring the conversion of [3H]benzo[alpha]pyrene to its various metabolites. Both mainstream as well as sidestream smoke exposure increased the pulmonary AHH activity by approximately 3-4-fold over room and sham control rats and mice, but not guinea pigs. The magnitude of AHH induction in sidestream- and mainstream-exposed animals was similar, even though the dose of smoke particulates received by rats and mice in sidestream-exposed groups was significantly less than the amount inhaled by their corresponding mainstream-exposed groups. The data suggest that like mainstream, the sidestream cigarette smoke is also capable of inducing pulmonary AHH in responsive animal species.     

Genetically mediated induction of aryl hydrocarbon hydroxylase activity in mice by polychlorinated dibenzofuran isomers and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Hepatic aryl hydrocarbon hydroxylase (AHH)-inducing potency of toxic polychlorinated aromatic hydrocarbons such as polychlorinated dibenzofurans (PCDFs), 3,4,5,3,4,5-hexachlorobiphenyl (HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in four inbred strains of mice with different phenotypes of Ah locus, i.e., AHH-responsive strains: C57BL/6N and AKR/Ms Qdj, and AHH-nonresponsive strains: DBA/2Cr Slc and Qdj; DDD. Eight individual PCDF isomers or TCDD were administered IP in doses of 30 g/kg; HCB was given in a dose of 120 g/kg. In AHH-nonresponsive strains of mice, only TCDD significantly induced hepatic AHH activity, while in AHH-responsive strains, 2,3,7,8-tetrachlorodibenzofuran(2,3,7,8-TCDF), 1,2,3,7,8-pentachlorodibenzofuran(1,2,3,7,8-PCDF) 2, 3, 4, 7, 8-pentachlorodibenzofuran (2, 3,4, 7, 8-PCDF), and TCDD significantly enhanced the enzyme activity, and the induced AHH activities with the three PCDF isomers were about 30–65% of those of TCDD. These results indicate that AHH responsiveness in mice segregates with the induction of AHH activity by PCDF isomers and may also segregate with the toxic potency of the isomers; i.e., toxic potencies of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF in AHH-responsive strains of mice may be much greater than those in AHH-nonresponsive strains of mice. Taking into account both the potent AHH inducibility and the high bioaccumulation of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF, these three PCDF isomers should be given greater attention with regard to environmental contamination.A part of this work was presented at the 41st annual meeting of the Japanese Society of Public Health, October 27–29, 1982, Fukuoka, Japan.     

Cytochrome P-450 and monooxygenase activity in hepatic microsomes from N-phenylimidazole-treated rats

Repeated administration of N-phenylimidazole (PI) to rats (3 daily doses of 200 mumol/kg/day) enhanced hepatic microsomal cytochrome P-450 levels (approx. 130%) and aminopyrine N-demethylase (APDM) and aniline p-hydroxylase (APH) activities (approx. 140%); aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were not enhanced over control values under similar conditions. Spectral studies with PI-induced microsomes indicated that although type II PI-binding characteristics were similar to those observed in controls, the 427 nm/455 nm absorbance ratio of the type III dihydrosafrole metabolite-cytochrome P-450 complex was lower than that in control microsomes. The results suggest that the inducing characteristics of PI bear some resemblance to those of phenobarbital (PB).     

A comparative study of polychlorinated dibenzofurans,polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin on aryl hydrocarbon hydroxylase inducing potency in rats

The aryl hydrocarbon hydroxylase (AHH) inducing potency of toxic chlorinated aromatic hydrocarbons such as polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in the young male Wistar rats. Alternatively, a technical PCDF mixture, 15 individual PCDF isomers or TCDD were administered i.p. in doses of 5 g/kg; a PCB mixture was given in a dose of 50 mg/kg. The order of AHH inducing ability was TCDD > PCDFs PCBs in kidney, lung, and liver. In the prostate, thymus, and spleen, only TCDD enhanced the AHH activity. The AHH inducibility in the lung and liver, induced by 15 pure PCDF isomers with varying chlorine substitutions was also examined. Only 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-tetra-CDF) and 2,3,4,7,8-pentachlorodibenzofurans (2,3,4,7,8-penta-CDF) significantly induced the hepatic AHH activity (4- and 2-fold, respectively), while eight PCDF isomers, including these two, significantly enhanced the pulmonary AHH activity (6- to 30-fold). Taking into account both the potent AHH inducibility and the high bioaccumulation of these compounds, 2,3,7,8-tetra- and 2,3,4,7,8-penta-CDF should be given due attention with regard to environmental-related factors and the possibility of involvement in the etiology of Yusho disease.A part of this work was presented at the 51st annual meeting of the Japanese Society for Hygiene, May 1–3, 1981, Sapporo, Japan and the 52nd annual meeting of the Japanese Society for Hygiene, March 29–31, 1982, Tokyo, Japan     

Small intestinal cytochromes P450.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

Extracts of airborne particulates collected at different locations in the Copenhagen area induce the expression of cytochrome P-450IA1

Acetone extracts of airborne particulates collected at different sites in the greater Copenhagen area were tested for their ability to induce the expression of cytochrome P-450IA1 RNA in a human breast cancer cell line, T47-D. The induction efficiency was expressed as an benz[a] anthracene equivalents, that is, the amount of benz[a]anthracene required to give the same level of induction. A significantly higher level of induction of P-450IA1 RNA was seen with samples collected on days with a smog alert. The inducibility of samples collected in rural areas was lower, but no significant difference in inducibility was found between samples collected in urban and suburban areas. Lack of correlation between the mutagenic activity in the Ames assay and the P-450IA1-inducing activity of the samples suggests that the complex mixture of compounds found in airborne particulates may have different biological activities in the two short-term test systems. Measurements of P-450IA1 inducibility provide a new, sensitive approach to assess the biological activity of material present in air pollution. The presence in airborne particulates of chemical compounds that induce cytochrome P-450IA1 an enzyme responsible for the metabolism of ubiquitous chemical carcinogens, suggests that the general environment may change an individual's response to the impact of exogenous chemicals, including the carcinogens present in cigarette smoke.     

The existence of the 4S polycyclic aromatic hydrocarbon-protein binding in 14-day-old chick embryo liver.

Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.     

Small Intestinal Cytochromes P450

Abstract

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally — being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics — with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid — aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly — iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on cytochrome P-450-dependent monooxygenase activities in rat hepatoma cells: induction of P-450I and suppression of P-450II

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytochrome P-450-dependent monooxygenase activities in several differentiated and dedifferentiated Reuber rat hepatoma cell lines using aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and aldrin epoxidase (AE) as test systems. The following results were obtained: (1) Exposure of cultures to 400 nM TPA for 18-24 h increased AHH activities in the differentiated lines 2sFou, H41IEC3/G- and Fao as well as in the dedifferentiated line 5L, 1.5-2.5-fold. The phorbol ester did not affect AHH activity in the dedifferentiated line H5. (2) EROD, a marker for P-450I, was induced by the phorbol ester to a similar degree as AHH. (3) A monoclonal antibody directed against P-450I strongly inhibited the AHH activity induced by TPA. (4) The onset of AHH or EROD induction by TPA was much later than that elicited by benz[a]anthracene. (6) In contrast to the induction of AHH and EROD, TPA decreased AE activity, a marker for P-450II, by about 50% in all the cell lines containing this monooxygenase activity. (7) The half-maximum-effect concentration of TPA for inducing or suppressing AHH and AE, respectively, was approximately 20 nM. (8) TPA did not interfere with AHH induction by benz[a]anthracene. However, the phorbol ester moderately decreased AHH induction and markedly suppressed AE induction by dexamethasone. The results indicate that TPA simultaneously induces P-450I and suppresses P-450II forms in rat hepatoma cells. P-450I induction by TPA in these cells did not appear to depend on their status of differentiation. Furthermore, the results suggest that the mechanism of P-450I induction by TPA differs from that elicited by polycyclic aromatic hydrocarbons or glucocorticoids.     

Studies on the induction of aryl hydrocarbon(benzo[a]pyrene) hydroxylase in Neurospora crassa, and its suppression by sodium selenite

Six fungal species were grown in the presence of benzo[a]pyrene (BP); four showed benzo[a]pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) activity. Penicillium sp. and Neurospora crassa metabolized BP to a limited extent. N. crassa AHH activity was induced by BP, the major product of metabolism being 3-hydroxy-BP. Both induction of AHH activity and metabolism of BP were suppressed by sodium selenite in the growth medium. Two polypeptides, unique to BP-grown cells, were revealed by two-dimensional electrophoretic separation of [35S]methionine-labelled proteins in N. crassa cell extracts. In selenium-grown cells the synthesis of BP-specific polypeptides was severely inhibited.