Metabolism Of Foreign Compounds Research Articles

Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor.

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-/- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice were reduced in size by 50 percent and showed bile duct fibrosis Ahr-/- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system.

Cell systems for use in studies on the relationship between foreign compound metabolism and toxicity.

Since the metabolism of most foreign compounds is predominantly controlled by hepatic in metabolism, isolated hepatocytes in most cases quite well predict the pattern of the overall metabolism of a given compound. Methods have been developed for cryopreserving isolated hepatocytes from man and other species with satisfactory maintenance of foreign compound metabolizing enzyme activities. The installation of a bank of cryopreserved hepatocytes from different species is possible and may be used for rational species extrapolation. It is necessary for some toxicological investigations to have hepatocytes which retain their differentiated status in culture for a sufficient time period. This might be achieved by co-culturing hepatocytes with diverse cell lines. However, from one cell line to the other differences in the pattern of stabilization of individual hepatocyte functions are found. In addition, questions on metabolic action of individual isoenzymes can also be addressed by the use of genetically engineered cell lines. All the in vitro systems mentioned, especially those which contain differentiated human cells or human isoenzymes are helpful in the rational species extrapolation of toxic effects from animal to man.

Interaction of Proteins with a Cytochrome P450 2B2 Gene Promoter: Identification of Two DNA Sequences That Bind Proteins That Are Enriched or Activated in Response to Phenobarbital

Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.

Role of human cytochrome P-450s in risk assessment and susceptibility to environmentally based disease.

Cytochromes P-450 (P-450s) are a large group of heme-containing proteins that carry out oxidation of numerous chemicals. In mammals, a limited number of P-450s are involved in metabolic pathways of steroid synthesis, while most of these enzymes are involved in metabolism of foreign compounds. The principal beneficial function of P-450s is to convert chemicals into derivatives that can be easily eliminated from the body. This generally occurs through P-450-mediated oxidations of hydrophobic substances followed by conjugation reactions. For many foreign compounds, P-450 metabolism results in production of "activated" metabolites that can cause cell death and gene mutations. During the past several years, it has become widely recognized that marked species differences occur among the foreign compound-metabolizing P-450s. In addition to this interspecies variability in metabolism, marked intraspecies variability, frequently referred to as drug oxidation polymorphisms, occurs in virtually all mammals examined to date. Based on these observations, it is necessary to develop new human P-450-based systems that can be used to study foreign compound metabolism in order to predict human risk. This is being accomplished by use of cDNA-directed expression in B lymphoblastoid cells. These cells can be used to predict how humans will metabolize a chemical and whether it will be metabolically activated to a toxic or mutagenic metabolite. To study human P-450 polymorphisms, polymerase chain reaction (PCR) assays have been developed for diagnosis of known mutant P-450 genes. Molecular probes are also being used to screen populations for levels of expression of carcinogen-activating P-450s in an effort to determine whether expression of certain P-450 forms is associated with increased risk for development of environmentally based disease.

Sexual differentiation and regulation of cytochrome P-450 CYP2C7.

The multigene family of proteins known as the cytochrome P-450-dependent monooxygenases play a central role in the metabolism of hormones and foreign compounds. As part of our studies into the function and regulation of these proteins we have isolated a little studied constitutively expressed isozyme CYP2C7 and have investigated its substrate specificity and mode of regulation. Interestingly the haem of this enzyme in its isolated form is almost 100% in the high spin state. The enzyme was active in the metabolism of a range of model resorufin substrates, but exhibits highest activity towards benzyloxyresorufin. Indeed, this isozyme appears to play a significant role in the metabolism of this substrate in microsomal samples from untreated male rats. Tissue distribution studies indicated that CYP2C7 was expressed in liver, kidney and possibly muscle tissue. Cytochrome P-450 CYP2C7 could not be significantly induced by any of a wide range of known modulators of cytochrome P-450 expression at the mRNA level, however some significant changes in protein expression were observed. Some of the agents used (e.g., diethylnitrosamine and carbon tetrachloride) caused a significant reduction in the expression of this protein. In agreement with other reports where mRNA levels were measured we found that the level of CYP2C7 protein expression was sexually differentiated. Female rats express two to three times the level found in males, the sex difference being reversible by hypophysectomy.

In vivo and in vitro models of the human colonic flora.

The study of colonic flora composition and metabolism presents considerable methodological problems. Attempts to circumvent these problems have led to the development of numerous in vitro and in vivo models to simulate the human colon and its microbial population. In terms of in vivo models, conventional laboratory animals have many limitations. Data of greater relevance to man can be obtained by using germ-free rodents associated with human colonic bacteria. The applications of such animals to studies of toxicity of chemicals and gastrointestinal infections are discussed. The advantages and disadvantages of the various in vitro systems for studying gut microflora and its metabolic activity (from simple static cultures to the more sophisticated continuous and semicontinuous flow models) are reviewed. The apparatus involved is described together with practical information on media, running conditions, and sampling. The bacteriological and metabolic criteria for establishing the similarity of the models to the in situ colonic flora are also discussed. The final sections of the review are devoted to the major applications (current and future) of the models, including fermentation studies on dietary fiber, metabolism of nutrients and foreign compounds (including carcinogens) in food, and the investigation of colonization resistance.

Control of constitutively-expressed developmentally-activated rat hepatic cytochrome P450 genes.

Cytochromes P450 (P450) collectively refer to a superfamily of heme-containing enzymes that use O2 and electrons from NADPH to insert a single atom of oxygen into any one of a large number of substrates. Two general classes of P450s exist; a relatively limited number of P450 forms, expressed in specialized tissues that are associated with pathways of steroidogenesis and a large number of forms responsible for metabolism of foreign compounds. Most of the latter P450s are expressed in liver, the primary site for metabolism of drugs, unusual dietary compounds and environmental pollutants. Numerous forms of P450 are expressed in liver of untreated animals and these are regulated quite differently. Both developmentally-programmed and sex-specific expressions have been observed. In this review, I will summarize recent findings on the mechanisms by which two P450 genes are regulated in livers of developing rats. The CYP2E1 gene is transcriptionally activated within a few hours after birth while the CYP2C6 gene is activated just prior to rats reaching puberty. These genes are under control of two transcription factors, HNF-1 alpha and DBP, respectively, that themselves are developmentally controlled.

Foreign compound metabolism capacity in man measured from metabolites of dietary caffeine.

Caffeine is sequentially metabolized by cytochrome P4501A2 (CYP1A2), N-acetyltransferase (NAT) and/or xanthine oxidase (XO). In the present study the activity of these three enzymes was estimated from ratios of the metabolites formed from dietary caffeine and excreted into the urine collected as spot samples. In the urine samples from 10 out of 377 subjects concentrations of caffeine metabolites were too low to allow reliable measurements of the ratios. In 335 healthy subjects the NAT activity showed a typically bimodal distribution with 47% fast acetylators and 53% slow acetylators, consistent with a Danish population. The ratios reflecting CYP1A2 and XO activities were log normal and normal distributed, respectively. In 103 non-smoking men and 90 non-smoking women the ratio of caffeine metabolites expressing CYP1A2 activity was 4.7 +/- 1.6 and 4.3 +/- 1.9 as compared to 7.8 +/- 2.5 and 7.3 +/- 3.0 in 31 male and 25 female subjects smoking 10 cigarettes/day or more respectively, verifying induction of CYP1A2 by tobacco (P less than 0.05), but minimal sex-related differences. In 12 non-smoking pregnant women and in 28 women using oral contraceptives the CYP1A2 ratio was 29 and 20% reduced respectively (P less than 0.05). In a multivariate analysis the only significant predictor of the XO ratio was the consumption of caffeine with an increase of 2% per cup of coffee or equivalent (P less than 0.05). In 23 healthy male subjects 30 days of vigorous exercise increased the CYP1A2 ratio by 70% and the XO ratio by 42% (P less than 0.05), but left the NAT ratio unchanged. In nine healthy volunteers daily ingestion of 500 g of broccoli for 10 days increased the CYP1A2 ratio by an average of 12% (P less than 0.05), compared to a control period with ingestion of an equivalent weight of non-cruciferous green vegetables. The ratios of metabolites from dietary caffeine in spot urine samples offer ethical, non-invasive and reliable estimates of CYP1A2, NAT and XO. These enzymes are highly relevant for the bioactivation of potentially toxic compounds and the formation of oxygen radicals. The method is applicable in large-scale epidemiological studies, allowing, for example, prospective testing of the relationship between these enzyme activities and the development of disease. Exercise may increase CYP1A2 activity to a magnitude corresponding to heavy smoking, as well as XO by mechanisms that remain to be clarified.

Glutathione S-transferase in intestine, liver and hepatic lesions of mummichog (Fundulus heteroclitus) from a creosote-contaminated environment

Cytosolic glutathione S-transferase (GSH transferase) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was elevated approximately three to four-fold in intestine and liver of mummichog (Fundulus heteroclitus) collected from a creosote-contaminated site in the Elizabeth River, Virginia. Intestinal GSH transferase activity at the most heavily contaminated site, at a moderately contaminated site and at a relatively clean site averaged 3.64, 2.83 and 1.11µmoles/min/mg respectively, while values for liver at these sites averaged 2.84, 1.75 and 0.93µmoles/min/mg. In addition, densitometric tracings of sodium dodecylsulfate-polyacrylamide gels of intestine and liver cytosol revealed a similar trend in the staining intensity of a 25.8 kD protein band, which lies within the molecular weight range of GSH transferase subunits. Activity in putative preneoplastic and neoplastic hepatic lesions of fish collected from the creosote-contaminated site was not significantly different from that of adjacent normal tissue. In the laboratory, dietary betanaphthoflavone (ßNF) treatment resulted in a three-fold increase in intestinal GSH transferase. Hepatic GSH transferase activity in the same fish was not affected by dietary ßNF although hepatic monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), was. The results of this study indicate a response of the intestinal detoxification system to environmental contaminants and supports previous studies on the importance of intestinal metabolism of foreign compounds. Further, our results indicate the trend towards elevated GSH transferase in liver of feral fish could not be attributed to a cancerous disease state in these fish but indicates chemical induction in this organ as well.

Difficulties in using animal data to predict pharmacological response in man

Variation among species in their response to xenobiotic agents depends upon two sets of factors: pharmacokinetic and pharmacodynamic. Pharmacokinetic factors, of which the rate of metabolic transformation is the most important, determine the concentration of the agent in plasma and tissues as a function of time after it is administered, while pharmacodynamic factors define the tissue response to a given concentration. Both are subject to considerable uncertainty when dealing with a new compound, so that it is not in general possible to predict reliably the response of human subjects to a new compound solely from studies on experimental animals. This uncertainty can be reduced substantially by understanding its mechanisms of action. Greater accuracy in predicting the metabolism of foreign compounds in man is also likely to be achieved by studies of the species variation of cytochrome P450s, and by the use of human hepatocytes and other cell lines to study xenobiotic metabolism.

Insights into deuterium isotope effects on the metabolism of steroid hormones and foreign compounds by cytochrome P-450 enzymes

Insights into deuterium isotope effects on the metabolism of steroid hormones and foreign compounds by cytochrome P-450 enzymes

Metronidazole and antipyrine metabolism in the rat: Clearance determination from one saliva sample

1. The applicability of a simple, non-invasive method for assessment of metronidazole and antipyrine metabolism in rats in vivo was investigated. 2. In 48 sample pairs of blood and pilocarpine-stimulated saliva from six rats the concentration of metronidazole was almost identical (r = 0.97). 3. In 26 rats the clearance could be determined from one sample without loss of precision and accuracy compared with conventional determinations (r = 0.99). If urine was collected for 24 h the fractional clearance representing each elimination pathway could be determined. 4. Pretreatment with phenobarbitone increased the fractional clearance of metronidazole by oxidation and glucuronidation 3.8-fold and 1.6-fold, respectively, whereas 3-methylcholanthrene pretreatment increased the rate of oxidation 10-fold and decreased the rate of glucuronidation 0.5-fold. 5. The clearance and fractional clearances of metronidazole and antipyrine administered in a mixture could be determined from the same saliva sample and urine collected for 24 h without drug-drug interactions. 6. Phenobarbitone pretreatment increased the formation rate of all metabolites of metronidazole and antipyrine administered in a mixture, whereas beta-naphthoflavone increased the formation rates of only the oxidative metronidazole metabolites, norantipyrine and 4-hydroxyantipyrine, but not metronidazole glucuronide or 3-hydroxymethylantipyrine. 7. A mixture of metronidazole and antipyrine and non-invasive sampling are recommendable for the study of the differential metabolism of foreign compounds in rats in vivo.

Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure.

High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons, but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. These rates are not obviously inducible by exposure to PAHs or PCBs. The lower rates of foreign compound metabolism contribute to higher pollutant residue levels in bivalve mollusks (clams, mussels, etc.) than in fish and may limit the involvement of some procarcinogens (requiring activation) in disease processes in invertebrates. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Metronidazole and Antipyrine as Probes for the Study of Foreign Compound Metabolism

The aim of the present work was to develop a tool for the study of the enzyme activities relevant for the biotransformation of foreign compounds, their elimination and/or activation to toxic substances. The activity of an enzyme may be assessed by the rate of metabolism of a preferably specific probe or model compound. The cytochrome P450'ies, the key enzymes for the elimination and/or activation of most foreign compounds, exist in multiple forms with variable substrate specificity and regulation. Some cytochrome P450'ies are under genetic control, whereas the activity of others is mainly regulated by the influence from factors in the environment. Only some of the cytochrome P450'ies are relevant for the formation of harmful metabolites. Thus, the activity of as many cytochrome P450 forms as possible should be assessable, preferably simultaneously. The present work evaluated metronidazole in a cocktail with antipyrine as a tool for the study of the regulation of foreign compound metabolism in the liver. The cytochrome P450 catalyzed metabolism of metronidazole and antipyrine was studied in humans and in isolated rat hepatocytes. In humans the influence of dose, route of administration, enzyme induction and inhibition and liver disease was investigated. Rats of either sex were studied with and without pretreatment with specific enzyme inducers and incubations included specific enzyme inhibitors. Evidence was provided that the oxidative formation of the five major metabolites, two from metronidazole and three from antipyrine, depends on different cytochrome P450'ies. In humans it was demonstrated that the clearance of metronidazole and antipyrine could be determined from the same saliva sample collected 16-24 hours after their oral administration and so could the clearance for formation of each metabolite if urine was collected for 48 hours. Thus, with the cocktail of metronidazole and antipyrine and simple non-invasive sampling the activity of five different cytochrome P450'ies can be assessed in vivo. In addition, metronidazole may also be used for assessment of the glucuronidation capacity although this is a minor pathway in man. Because the variation within subjects is much less than between them, the cocktail test is particularly suited for paired designs with measurements before and after an environmental change and the subjects serving as their own control. The metronidazole/antipyrine cocktail may have many applications in the study of the regulation of foreign compound metabolism in man and in animals, in vivo and in vitro.

Pulmonary metabolism of foreign compounds: its role in metabolic activation.

The lung has the potential of metabolizing many foreign chemicals to a vast array of metabolites with different pharmacological and toxicological properties. Because many chemicals require metabolic activation in order to exert their toxicity, the cellular distribution of the drug-metabolizing enzymes in a heterogeneous tissue, such as the lung, and the balance of metabolic activation and deactivation pathways in any particular cell are key factors in determining the cellular specificity of many pulmonary toxins. Environmental factors such as air pollution, cigarette smoking, and diet markedly affect the pulmonary metabolism of some chemicals and, thereby, possibly affect their toxicity.

Pulmonary Metabolism of Foreign Compounds: Its Role in Metabolic Activation

Pulmonary Metabolism of Foreign Compounds: Its Role in Metabolic Activation

Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers

1. Polychlorinated biphenyls (PCB) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB-contaminated lipid components of food chains. 2. Commercial mixtures of PCB induce, in hepatic microsomal membranes in vivo, a variety of different forms of the cytochrome P-450 components of enzyme systems involved in the metabolism of drugs and other xenobiotics, and can also induce the proliferation of this membrane. Since these microsomal enzyme systems share a number of the requirements of microsomal fatty acid desaturases, we have investigated whether the induction by PCB in vivo of cytochrome-P-450-linked enzymes in the proliferating hepatic microsomal membrane of the pigeon and the rat is accompanied by increased proportions of polyunsaturated fatty acids in this membrane. 3. The most striking changes observed 120 h after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body mass were 2.2-fold and 1.6-fold increases, respectively, in the proportion of arachidonic acid in the hepatic microsomal membrane. When the effects of this treatment on the proliferation of this membrane and increase in liver mass are taken into account, the amount of arachidonic acid in the total microsomal membrane of pigeon and rat livers increased 6.7-fold and 1.9-fold, respectively. 4. These changes were accompanied by very significant increases in pigeons and rats of the concentration of hepatic microsomal cytochrome P-450, and in the activity in microsomal protein of a wide range of cytochrome P-450-dependent enzyme involved in the metabolism of drugs and other xenobiotics. 5. This effect of PCB, of increasing in vivo the degree of unsaturation of fatty acids of hepatic microsomal membrane, appears to be a novel finding, and does not seem to have been investigated for other drugs and xenobiotics. Preliminary results have shown that the effect is accompanied by substantial increases in the total activity of delta 6 and delta 5 microsomal fatty acid desaturases converting 18:2 (9, 12) (linoleic acid) to 20:4 (5, 8, 11, 14) (arachidonic acid) [Borlakoglu, J.T., Dils, R.R., Edwards-Webb, J.D. & Walker, C.H. (1988) Biochem. Soc. Trans. 16, 1072]. 6. It is postulated that there is a significant link between increased fatty acid desaturation and the induction of cytochrome-P-450-linked enzymes, and this is discussed in terms of the mechanisms involved in the metabolism of foreign compounds.

Variations in the response of cell lines to metabolism-mediated toxicity

One major criticism of cytotoxicity tests is the negligible capacity for metabolism of foreign compounds, which exists in most cell lines. We have investigated the use of an exogenous metabolizing system, comprising 9000 g supernatant of liver from Aroclor 1254-pretreated rats (S-9) and an NADPH regenerating system, in the kenacid blue test. BCL-D1 cells (a human embryonic lung finite cell line) appeared to be sensitive to the toxicity of active metabolites of several model toxins. Other cell lines appeared to be less sensitive but the effects varied with the cell line and the test compound. These differences may, in part, be due to differences in the intrinsic ability of different cell lines to detoxify the active metabolites. It is therefore possible to use this type of test system for mechanistic studies but results with unknown compounds should be treated with caution.

Effects of Age, Adrenocorticotropin, and Dexamethasone on a Male-Specific Cytochrome P450 Localized in the Inner Zone of the Guinea Pig Adrenal*

Guinea pig adrenal microsomes have a capacity for xenobiotic metabolism which is localized to the inner zone, greater in adult males then females, greater in older than in younger males, and suppressed by ACTH. In this paper we show that a cytochrome P450 distinct from P450(21) and P450(17) alpha, which is localized to the inner zone, is male specific, increases with age, and is suppressed by ACTH. This is the first report of a sex-dependent cytochrome P450 in the adrenal. It is immunochemically related to the two members of the P450I family, P450 c and d, neither of which has been reported to be hormonally regulated. The correlation of this cytochrome P450 with the ability of the microsomes to metabolize ethylmorphine suggests that it accounts for this capacity. Whether metabolism of foreign compounds and/or male-specific steroid hydroxylation are its functions in vivo remains to be determined.

A cytochrome P450 immunochemically related to P450c,d (P450I) localized to the smooth microsomes and inner zone of the guinea pig adrenal.

In addition to their capacity for steroid synthesis, guinea pig adrenal microsomes have a well documented ability to metabolize foreign compounds. The capacity for metabolism of foreign compounds is localized to the smooth endoplasmic reticulum-filled cells of the inner zone. However, it has not been clear whether they possess cytochrome P450(s) specific for this function, distinct from the two known steroid hydroxylases, P450(21) and P450(17)alpha. Multiple prominent protein bands in the mol wt range of known cytochrome P450s are seen on sodium dodecyl sulfate gels of guinea pig adrenal microsomes. Most are more intense in smooth microsomes, where the concentration of cytochrome P450 is highest. However, one band (52K) appears unique to the smooth microsomes. This band is also characteristic of microsomes obtained from the inner zone. This protein and two others (54K and 50K) are concentrated in the membrane pellet after carbonate treatment of the microsomes, indicating that they are integral membrane proteins. All three decrease in intensity after treatment of the animals with spironolactone, a compound known to cause depletion of adrenal cytochrome P450s. On Western blots of microsomal proteins the 54K and 50K proteins react with antibodies specific for P450(17) alpha and P450(21), respectively. The 52K protein, characteristic of the smooth microsomes and inner zone, does not react with anti-P450(21) or anti-P450(17) alpha, but does react with polyclonal antibody raised against microsomal cytochrome P450s induced by methylcholanthrene in rat liver (P450c,d). These results suggest that there is at least one additional cytochrome P450 in adrenal microsomes which is immunochemically distinct from P450(21) and P450(17) alpha. Its localization to the smooth microsomes and inner zone microsomes correlates with the high activity for ethylmorphine metabolism detected in these fractions. This suggests that this cytochrome P450, which is immunochemically related to members of the P450I subfamily, may be associated with the ability of guinea pig adrenal microsomes to metabolize foreign compounds.