Toxicological implications of enzymatic control of reactive metabolites

Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.     

Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasoneinduced rat liver microsomes

1. Rapamycin is metabolically transformed in rat liver microsomes to 3,4- and 5,6- dihydrodiol metabolites under the influence of the cytochrome P-450 mixed function oxygenase system. These metabolites were produced from dexamethasone-induced as well as from non-induced rat liver microsomes. The comparison of the ion spray mass spectra of the 5,6-dihydrodiol with the 3,4-dihydrodiol of rapamycin shows clearly that dihydrodiols were formed in two distinct positions of rapamycin. 2. FAB mass spectra as well as electrospray mass spectra of two additional peaks isolated from the same chromatographic run confirm the presence of a 3,4-dihydrodiol metabolite of rapamycin as also strongly suggested by UV spectra.Hplc reinjection of each individualpeak always resultedinchromatograms showing a combinationof thesame three peaks and therefore are to be considered as tautomers of the 3,4-dihydrodiol of rapamycin. 3. These tautomeric conformations were found to have no immunosuppressive potency, most probably due to important structural and stereochemical modifications of the rapamycin binding domain to the binding protein (FKBP-12) and or to important metabolic structural modifications of rapamycin effector domain.     

Toxicological significance of mechanism-based inactivation of cytochrome p450 enzymes by drugs

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine.Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.     

临床生化指标在新药临床前研究中的毒理学意义

临床生化指标主要用来检测碳水化合物、脂类、蛋白质、尿液、肝胆、心血管、肌肉、胃肠道系统的代谢。临床检验的个体或汇总数据要在病理报告中分析并在实验报告中述及。阐明了临床生化指标在药物临床前研究中的毒理学和生物学意义,从而考察和反映了临床生化指标对动物机体总的代谢、营养和健康状态的影响,同时为建立和定期更新实验室各实验动物临床检验的背景值提供了参考与比较。     

Absolute configurations of the dihydrodiol metabolites of 5,5-diphenylhydantoin (phenytoin) from rat, dog, and human urine

Dihydrodiol metabolites (DHD) of phenytoin (PHT) have been extracted from the urine of rats and dogs, and from that of a patient on chronic PHT therapy. The crystalline rat DHD was dehydrated to give a mixture of (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin and (S)-5-(3-hydroxyphenyl)-5-phenylhydantoin. The absolute configuration of C5 of the hydantoin ring of the DHD can accordingly be assigned as (S). Circular dichroism studies allowed further assignment of absolute configuration to the crystalline rat DHD, the metabolite being identified as (5S)-5-[(3R,4R)-3,4-dihydroxy-1,5-cyclohexadien-1-yl]-5-phenylhydantoin, (5S)-DHD. The human DHD metabolite was identical with the (5S)-DHD. The DHD from dog urine was found to be a mixture of diastereoisomers; the major component was identified as (5R)-5-[(3R,4R)-3,4-dihydroxy-1,5-cyclohexadien-1-yl]-5-phenylhydantoin, (5R)-DHD, and the minor diastereoisomer was identified as (5S)-DHD. The ratio (5R)-DHD/(5S)-DHD was approximately 2. A comparison of the absolute configurations of phenolic metabolites of PHT and of the DHD's offered a basis for speculation as to stereochemical aspects of PHT metabolism in the rat, dog, and man.     

Biologically active dihydrodiol metabolites of polycyclic aromatic hydrocarbons structurally related to the potent carcinogenic hydrocarbon 7,12-dimethylbenz[a]anthracene

Syntheses of the trans-dihydrodiol derivatives implicated as the proximate carcinogenic metabolites of the polycyclic hydrocarbons cholanthrene, 6-methylcholanthrene, benz[a]anthracene, and 7- and 12-methylbenz[a]anthracene are described. These compounds are useful models for research to determine the molecular basis of the strong enhancement of carcinogenicity consequent upon methyl substitution in nonbenzo bay molecular sites and meso regions of polycyclic hydrocarbons. Synthesis of the bay region anti-diol epoxide derivative of cholanthrene, its putative ultimate carcinogenic metabolite, is also described. Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin. The most active member of the series is the dihydrodiol derivative of 6-methylcholanthrene, which contains a bay region methyl group. The ability of the dihydrodiols 3a-c and the trans-3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene (3d) to induce chromosomal aberrations in rat bone marrow cells was also examined. The observed order of activity was 3d greater than 3c greater than 3b greater than 3a. These findings are consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.     

Toxicological significance of hepatic responses to furylfuramide(AF-2) in the rat

In male rats fed furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2] at a dietary level of 0.1%, a marked increase in the liver weight was observed with a concomitant reduction of microsomal mixed-function oxidase activity. To clarify the significance of these hepatic responses to furylfuramide, a series of biochemical parameters that reflect hepatotoxicity was measured. The most significant elevations were seen in hepatic glycogen and glucose 6-phosphate dehydrogenase levels over 29-day feeding interval. A marked decrease was observed in mixed-function oxidases and glucose 6-phosphatase activities. Moderate decreases were seen in several other parameters, especially in the early phase of feeding. Measurement of serum parameters showed significant elevation in cholesterol and a transient increase in serum glutamic pyruvic transminase. A comparative study demonstrated small differences between furylfuramide and carbon tetrachloride in their effects on hepatic parameters. The furylfuramide-induced changes appear to be reversible within 14 days after withdrawal of furylfuramide from the diet. Reduction of mixed-function oxidase activity by furylfuramide might be caused partly by the elevation of heme degrading enzyme. The results indicate a dysfunction of drug-metabolizing activity and acute and slight toxication of the liver by furylfuramide.     

Toxicological information series,I: Toxicological information

This paper presents a brief history of the evolution of toxicological information in the United States since 1966 when concern over the hazards of the ubiquitous chemicals in the environment was translated into recommendations for action by The Panel on the Handling of Toxicological Information of the President's Science Advisory Committee. It describes some of the data bases that were developed as a result of these recommendations and introduces a series of papers that discuss toxicology information resources, their content, and their accessibility. The series is a project of the Information Handling Committee of the Society of Toxicology. Papers II–V of this series will be published in subsequent issues of Fundamental and Applied Toxicology.     

Toxicological screening

Well-defined dose- and time-related toxic effects of chemicals can often be detected using simple tests with small numbers of animals. The strategies for the establishment of toxicological screening tests are discussed. The most important steps are the definition of the targets, the selection of the methods, and the setting of the test criteria. Screening tests must then be validated with standard reference chemicals, and the test criteria must be adjusted so that the standards can be detected regularly. Maximal flexibility is allowed in the design of the tests. For evaluation, the results obtained in treated animals can be compared with those of controls, using conventional concepts of biostatistics. It is also possible to base the evaluation on preset test criteria. Toxicological screening tests do not replace conventional safety studies, but they help in selecting the most promising candidates in a series of related chemicals and in establishing priorities for further testing. As an example, a screening for the hemolytic effects of chemicals is presented.     

Toxicological Information

Toxicological Information. COSMIDES, G. J. (1990). Fundam. Appl.Toxicol. 14, 439–443. This paper presents a brief historyof the evolution of toxicological information in the UnitedStates since 1966 when concern over the hazards of the ubiquitouschemicals in the environment was translated into recommendationsfor action by The Panel on the Handling of Toxicological Informationof the President's Science Advisory Committee. It describessome of the data bases that were developed as a result of theserecommendations and introduces a series of papers that discusstoxicology information resources, their content, and their accessibility.The series is a project of the Information Handling Committeeof the Society of Toxicology. Papers II–V of this serieswill be published in subsequent issues of Fundamental and AppliedToxicology.     

Toxicological methods

Toxicity protocols intended to cover several countries may be complex. The long-term study has changed relatively little in 20 years, but has been refined through, e.g., routine ophthalmoscopy. Closer chemical control throughout a study is advocated. Dosage form, route and frequency of administration all affect toxicity. “Effect level” and “maximum tolerated dose” remain valuable concepts. In certain instances, experimental animals may be more sensitive than man. Toxicological pathology is comparative rather than diagnostic, and extrapolation to man of tumorigenicity may be difficult. Tetratogenicity is exerted through a susceptible genetic locus.     

Metabolism of 7,12-dimethylbenz(a)anthracene. Correlation of dihydrodiol and phenol metabolites with organ susceptibility to carcinogenesis in Sprague-Dawley and long-Evans rats

The biotransformation of DMBA in uninduced and induced 10,000g organ supernatants derived from female Sprague-Dawley and Long-Evans rats, which are respectively sensitive and resistant to DMBA-induced carcinogenesis, was studied. Qualitative and quantitative differences in the metabolism of DMBA are summarized as a function of organ (liver, lung, kidney, and mammary) and strain and oxidative enzyme induction protocol utilized and are attributed to the differential induction of various cytochrome P-450s. The percent production of 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (DMBA-3,4-DHD) and phenol metabolites in 10,000g organ supernatants from beta-napthoflavone induced animals correlated with organ susceptibility to DMBA-induced carcinogenesis in both SD and LE rat strains. A negative correlation was observed between 8,9-DHD production and reported sensitivity to specific organ tumorigenesis, but no other relationship was observed with other metabolites (7- and 12-hydroxymethyl, K-region DHD, and 10,11-DHDs). Similar DMBA metabolite hplc profiles were observed in the same organ S-10 fractions isolated from either SD or LE rats. Thus, the organotropic, but not the strain, selectivity of DMBA-induced carcinogenicity appears to be attributed in part to metabolic activation.