Werner Lilienblum

Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH)

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using “Integrated Testing Strategies” (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activities by various inducing agents

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.

Functional heterogeneity of UDP-glucuronyltransferase in rat tissues

Abstract Tissue distribution of UDP-glucuronyltransferase was investigated using two substrate groups which were shown to be conjugated by two different forms of this enzyme in previous studies with rat liver. These enzyme forms were found to be differentially inducible by 3-methylcholanthrene (form 1) and phenobarbital (form 2). Group 1 substrates (conjugated by form 1) include 1-naphthol, N -hydroxy-2-naphthylamine and 3-hydroxybenzo[ a ]pyrene; group 2 substrates (conjugated by form 2) comprise 4-hydroxybiphenyl, morphine and chloramphenicol. Group 1 substrates are conjugated in a number of tissues, for example, liver, kidney, small intestinal mucosa, lung, skin, testes and spleen. However, conjugation of group 2 substrates is detectable only in liver and intestine to an appreciable extent. It is concluded that enzyme(s) efficient in the conjugation of group 1 substrates is ubiquitous in the investigated organs, whilst only liver and intestine possess enzyme(s) efficient in the conjugation of group 2 substrates. In contrast to 3-hydroxybenzo[ a ]pyrene, benzo[ a ]pyrene 7,8-dihydrodiol cannot be clearly associated with only one of the 2 substrate groups. Glucuronidation of benzo[ a ]pyrene 7.8-dihydrodiol is enhanced by both phenobarbital and 3-methylcholanthrene in liver. Conjugation of the dihydrodiol is detectable in all tissues examined. However, enzyme activity towards the dihydrodiol is much lower than that towards 3-hydroxybenzo[ a ]pyrene. It is disproportionately low with skin microsomes.

The role of conjugation reactions in detoxication

Abstract(1)The role of conjugating enzymes is best understood by looking at the interaction between phase I (mostly cytochromes P-450) and phase II (conjugation) enzymes of drug metabolism. A balance between phase I and II enzymes of detoxication largely determines the disposition to drug toxicity. Reactive electrophilic metabolites, generated by phase I enzymes, are often controlled by GSH-tansferases, whereas nucleophilic metabolites such as phenols are controlled by UDP-glucuronosyltransferases (GT) and sulfotransferases. It is more and more recognized that the control of the more stable and more abundant nucleophiles is as important as the control of electrophiles, since the former can be readily converted to electrophiles. For example, phenols and quinols can undergo quinone/quinol redox-cycles with the generation of reactive oxygen species. In the case of benzo(a)pyrene-3,6-quinol toxicity can be prevented by glucuronidation.(2)Conjugating enzymes consist of families of isoenzymes with distinct but overlapping substrate specificity. Rather than dealing with individual isoenzymes, adaptive programs are emphasized by which gene expression of a battery of phase I and II enzymes is turned on by certain types of inducing agents. Mechanistically best known is the program turned on by 3-methylcholanthrene-type inducers which includes enhanced synthesis of certain isoenzymes of cytochrom P-450, GT and probably GSH-transferase. The program may adapt the organism to efficiently detoxify and eliminate aromatic compounds such as benzo(a)pyrene. Evidence is presented that this program exists in both rodents and humans.(3)The balance between phase I and II enzymes is permanently altered after initiation of hepatocarcinogenesis. Cytochromes P-450 are decreased both in liver foci of altered hepatocytes and nodules, whereas GTs and GSH-transferases are increased. The altered enzyme pattern is consistent with increased toxin resistance of initiated hepatocytes. This toxin-resistance phenotype leads to selective growth of initiated hepatocytes during continuous exposure to carcinogens and may thus facilitate the evolution of cancer cells.

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide.

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.

Functional heterogeneity of UDP-glucuronosyltransferase activities in C57BL/6 and DBA/2 mice.

Functional heterogeneity of liver microsomal UDP-glucuronosyltransferase activities towards 1-naphthol, 4-methylumbelliferone or 3-hydroxybenzo(a)pyrene (UDP-GT1 activities) and morphine or 4-hydroxybiphenyl (UDP-GT2 activities) was studied in two inbred strains of mice which are genetically responsive (C57BL/6) or non-responsive (DBA/2) to 3-methylcholanthrene-induction of drug metabolizing enzymes. 3-Methylcholanthrene preferentially induced UDP-GT1 activities in C57BL/6 mice. Phenobarbital, however, at low doses (50 mg/kg), selectively induced UDP-GT2 activities. Higher doses of phenobarbital (80 mg/kg) induced both UDP-GT1 and UDP-GT2 activities. In DBA/2 mice 3-methylcholanthrene-induction of UDP-glucuronosyltransferase activities was not detectable whereas enzyme induction by phenobarbital appeared to be unimpaired. UDP-GT1 activities were ubiquitously detectable in mouse tissues whereas appreciable UDP-GT2 activities were only found in liver and small intestinal mucosa. UDP-GT1 (1-naphthol as substrate) was not inhibited by morphine suggesting different active sites for the conjugation of these substrates. The results suggest the presence of at least two functionally different forms of UDP-glucuronosyltransferase in mice. In conjunction with the results of Owens (J. biol. Chem. 252, 2827 (1977)) it is evident that one of these enzyme forms is regulated by the Ah locus.

N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.

Induction of rat hepatic UDP-glucuronosyltransferases by dietary ethoxyquin

SummaryDietary administration of 0.5% ethoxyquin markedly enhanced rat hepatic UDP-glucuronosyltransferase activities. Both 3-methylcholanthrene- and phenobarbital-inducible glucuronidation reactions were stimulated by the antioxidant. In contrast, phenobarbital-inducible bilirubin glucuronidation was not affected by ethoxyquin.

Conversion of benzo[a]pyrene-3,6-quinone to quinol glucuronides with rat liver microsomes or purified nadph-cytochrome c reductase and UDP-glucuronosyltransferase

Polycyclic aromatic hydrocarbons such as benzo[alpyrene (BP) exert their toxic effects after metabolic activation. A delicate balance between activating and detoxifying enzyme reactions may be decisive for the accumulation of reactive intermediates in cells and may explain in part the organ specificity of toxic effects. UDP-glucuronosyltransferase (CT, EC 2.4. 1.17) is one of the enzymes conjugating intermediary phenols and dihydrodiols of BP [l-3], thus preventing direct cytotoxicity of these intermediates and recycling to ultimate cytotoxins, mutagens, and carcinogens [4]. BP quinones, a third major class of primary metabolites, can be readily conjugated with glucuronic acid after reduction to their quinols [5,6]. BP quinones may cause cytotoxicity in many ways, for example, after reduction to semiquinones or by oxidation-reduction cycles with continuous production of HzOz, 0, and OH’ [7]. In addition quinones strongly inhibit BP oxidation [6]. When inhibitory quinones are removed by stimulation of conjugation of the corresponding quinols, BP monooxygenase activity is increased [6,8]. CT probably consists of a family of closely related but functionally heterogeneous enzyme forms. Two forms are differentially inducible in rat liver by inducing agents such as 3-methylcholanthrene (MC) or phenobarbital [9,10]. They can be separated and purified to apparent homogeneity [ 1 l] and show different developmental patterns [ 121 and tissue distributions [3]. The methylcholanthrene-inducible form of GT is operationally called GT1 [3]. All enzyme forms are latent in the intact microsomal membrane. They can be activated by UDP-N-acetylglucosamine, which is probably a physiological activator, and can be fully activated by various alterations of the membrane structure, e.g., by the addition of detergents [ 131. To be able to distinguish induction from activation, enzyme induction is usually studied in the fully activated state. Because of the complexity of GT activities it was necessary to investigate which form of GT is responsible for the conjugation of BP quinols. We demonstrate that isolated GT1 and NADPH-cytochrome c reductase from rat liver, both purified to apparent homogeneity, readily form BP-3,6-quinol glucuronides from BP-3,6-quinone. Using a sensitive fluorimetric assay for the detection of BP-3,6quinol glucuronides, rates of conversion obtained with the purified enzymes are compared with those determined with the membrane-bound enzymes from liver microsomes.

Formation of pentachlorophenol glucuronide in rat and human liver microsomes

Abstract Pentachlorophenol (PCP) ∗ is a widely used fungicide which has been increasingly found as an environmental contaminant. Toxicological properties and pharmacokinetics of this substance have recently been reviewed [1]. The major part of an oral dose was found in urine as free PCP [2–5]. In rats, 6–16% of the dose were recovered from urine as PCP glucuronide [3, 5]. In pilot experiments, PCP glucuronide was found to be unstable at pH 5, the pH normally used when glucuronides are identified by hydrolysis with β-glucuronidase. Therefore we investigated PCP glucuronide synthesis in rat and human liver microsomes and its stability at urinary pH. In the rat liver model of differential induction [6–11] it was found that glucuronidation of PCP is neither inducible by 3-methyl-cholanthrene nor by phenobarbital. Based on the appreciable rate of PCP conjugation in liver and the instability of the conjugate at the urinary pH it is concluded that the determination of PCP glucuronide in urine leads to an underestimation of the amount of this conjugate originally excreted via the kidney.