Multiple activation of chloroform in kidney microsomes from male and female DBA/2J mice

Ade, Paola; Guastadisegni, Cecilia; Testai, Emanuela; Vittozzi, Luciano

doi:10.1002/jbt.2570090603

Cited by 16 publications

(5 citation statements)

References 23 publications

(21 reference statements)

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“…1). The relative ratio between the two pathways depends on the oxygen partial pressure, on chloroform concentration and is specie-, organ-and gender-specific (Ade et al, 1994;Gemma et al, 1996;Vittozzi et al, 2000).…”

mentioning

confidence: 99%

Metabolism of Chloroform in the Human Liver and Identification of the Competent P450s

Gemma¹,

Vittozzi²,

Testai³

2003

Drug Metab Dispos

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ABSTRACT:The oxidative and reductive cytochrome P450 (P450)-mediated chloroform bioactivation has been investigated in human liver microsomes (HLM), and the role of human P450s have been defined by integrating results from several experimental approaches: cDNA-expressed P450s, selective chemical inhibitors and specific antibodies, correlation studies in a panel of phenotyped HLM. HLM bioactivated CHCl 3 both oxidatively and reductively. Oxidative reaction was characterized by two components, suggesting multiple P450 involvement. The high affinity process was catalyzed by CYP2E1, as clearly indicated by kinetic studies, correlation with chlorzoxazone 6-hydroxylation (r ‫؍‬ 0.837; p < 0.001), and inhibition by monoclonal antihuman CYP2E1 and 4-methylpyrazole. The low affinity phase of oxidative metabolism was essentially catalyzed by CYP2A6. This conclusion was supported by the correlation with coumarin 7-hydroxylase (r ‫؍‬ 0.777; p < 0.01), inhibition by coumarin and by the specific antibody, in addition to results with heterologously expressed P450s. Chloroform oxidation was poorly dependent on pO 2 , whereas the reductive metabolism was highly inhibited by O 2 . The production of dichloromethyl radical was significant only at CHCl 3 concentration >1 mM, increasing linearly with substrate concentration. CYP2E1 was the primary enzyme involved in the reductive reaction, as univocally indicated by all the different approaches. The reductive pathway seems to be scarcely relevant in the human liver, since it is active only at high substrate concentrations, and in strictly anaerobic conditions. The role of human CYP2E1 in CHCl 3 metabolism at low levels, typical of actual human exposure, provides insight into the molecular basis for eventual difference in susceptibility to chloroform-induced effects due to either genetic, pathophysiological, or environmental factors.

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mentioning

confidence: 99%

Metabolism of Chloroform in the Human Liver and Identification of the Competent P450s

Gemma¹,

Vittozzi²,

Testai³

2003

Drug Metab Dispos

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“…One approach to refining a genotoxicity assay has been to change the metabolising system with regard to the type of system, organ, sex, species, and, in particular, to permit the use of human-derived sources of enzymes (29). In the case of kidney-specific toxicity of TRE discussed earlier, the bacterial mutagenicity of this chemical was only apparent when microsomes from the kidney were used (58).…”

Section: Subcellular Metabolising Systemsmentioning

confidence: 98%

“…Interstrain differences in biotransformation can give rise to discrepant toxicity data. Variation in the renal toxicity of chloroform is another example of the effects of such differences in biotransformation (58).…”

Section: Species Differencesmentioning

confidence: 99%

Metabolism: A Bottleneck inIn VitroToxicological Test Development

et al. 2006

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This is the 54th report of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). The main objective of ECVAM, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences, and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and of opportunities for the possible incorporation of alternative methods into regulatory procedures. It was decided that this would be best achieved through a programme of ECVAM workshops, each addressing a specific topic, and at which selected groups of independent international experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward.A workshop on Metabolism: a bottleneck in in vitro toxicological test development, was held at

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“…Livers and kidneys were removed rapidly and microsomes were prepared as described previously (Testai & Vittozzi, 1986;Ade et al, 1994 CHCl 3 injection, rats were transferred into all-glass metabolic cages (Metabowls MK III, Jencons Scientific) supplied with a constant dry and CO 2 -free airflow of 500 ml min −1 by a vacuum pump. The outgoing air bubbled in a series of three traps; two of them contained toluene for 14 CHCl 3 trapping (300 ml each) and the third was filled with 300 ml of Lumasorb II to collect expired 14 CO 2 .…”

Section: Animals Microsomal Preparation and In Vivo Treatmentmentioning

confidence: 99%

“…The use of the results from such studies in F344 rats for the interpretation of bioassay results with OM rats requires that similarities in the key steps of chloroform metabolism be ascertained. Indeed, the complex bioactivation of chloroform (Testai et al, 1990De Biasi et al, 1992;Ade et al, 1994;Gemma et al, 1996a;Vittozzi et al, 2000) may produce a variety of primary and secondary metabolites with different chemical reactivity and cytotoxicity (De Biasi et al, 1992;Ade et al, 1994;Gemma et al, 1996aGemma et al, , 1996bRossi et al, 1999;Vittozzi et al, 2000;Di Consiglio et al, 2001). These oxidative and reductive metabolic pathways are found in vitro in several combinations in the livers and kidneys of B6C3F1 mice and in Sprague-Dawley and OM rats (Gemma et al, 1996a;Vittozzi et al, 2000).…”

Section: Introductionmentioning

confidence: 97%

Bioactivation, toxicokinetics and acute effects of chloroform in fisher 344 and Osborne Mendel Male rats

Gemma

Testai

Chieco

et al. 2004

J of Applied Toxicology

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Chloroform has been regarded as a renal carcinogen, based on results obtained with Osborne Mendel (OM) rats. Fisher 344 (F344) rats, considered representative of OM rats on the basis of comparable acute toxic effects, have been used in most of the studies aimed to elucidate the mechanisms of kidney tumour induction. In the present work, in vitro and in vivo chloroform bioactivation in the liver and kidney of F344 and OM rats has been reported, as well as additional toxicokinetics and acute toxicity information. Complete similarity of chloroform metabolism and toxicokinetics was evidenced in the two rat strains. Chloroform metabolism was fully saturated at the OM rat bioassay doses (90-180 mg kg(-1) body wt.), working at a maximal rate of 40-50 micro mol (14)CO(2) expired kg(-1) h(-1). No acute hepatotoxicity, nephrotoxicity or consequent cell proliferation was evidenced at 180 mg kg(-1) body wt. chloroform. In the rat liver, phosgene was confirmed as the major metabolite. Renal microsomes from both F344 and OM rats in vitro were unable to produce any oxidative metabolite; at variance, adducts due to oxidative and reductive metabolites were detected in vivo. Our results indicated the presence in the rat kidney of electrophilic metabolites other than phosgene, representing either oxidative metabolites formed elsewhere and sufficiently stable to be transported to the kidney or electrophilic metabolites secondary to the formation of reductive radicals. Therefore, the rat kidney represents a suitable model to study the toxicological effects, including genotoxicity, of chloroform metabolites in the absence of cytotoxic effects produced by phosgene formed in situ.

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Multiple activation of chloroform in kidney microsomes from male and female DBA/2J mice

Cited by 16 publications

References 23 publications

Metabolism of Chloroform in the Human Liver and Identification of the Competent P450s

Metabolism of Chloroform in the Human Liver and Identification of the Competent P450s

Metabolism: A Bottleneck inIn VitroToxicological Test Development

Bioactivation, toxicokinetics and acute effects of chloroform in fisher 344 and Osborne Mendel Male rats

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