The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers

Wortelboer, Heleen M.; Kruif, C.A. de; Iersel, A.A.J. van; Falke, H.E.; Noordhoek, J.; Blaauboer, Bas J.

doi:10.1016/0006-2952(90)90095-3

Cited by 153 publications

(61 citation statements)

References 28 publications

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“…The major metabolite formed was androstenedione both in cryopreserved and non-cryopreserved hepatocytes, and this is consistent with what we and others have previously reported for cultured hepatocytes Kern et al, 1997). The formation of the 6--hydroxy and 7--hydroxy-testosterone metabolites was the most labile after freezing the cells, and they represent the activity of the CYP 2A1 family of P450 isoenzymes Wortelboer et al, 1990;Hengstler et al, 2000). CYP2C11 is the major form of P450 in the male rat liver (Schenkman et al, 1989), and it is solely responsible for the formation of 2--hydroxytestosterone, and along with CYP2B1 catalyses the 16--hydroxylation and the formation of androstenedione.…”

Section: 3supporting

confidence: 90%

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Cryopreservation of Hepatocytes for Bioartificial Liver Devices

Stevenson

Grant

2005

Bioreactors for Tissue Engineering

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Section: 3supporting

confidence: 90%

“…These are the main ATP producing pathways in hepatocytes, and lack of energy to activate the UDP-glucuronic acid donor for glucuronidation reactions may explain why this conjugation reaction is more labile than phase I cytochrome P450 reactions in post-thaw cryopreserved cells. (Hengstler et al, 2000;Wortelboer et al, 1990). …”

Section: 3mentioning

confidence: 99%

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

Stevenson

Grant

2005

Bioreactors for Tissue Engineering

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“…The S9-fraction was obtained from Aroclor-1254 treated-male rats (U:WU(CPB) Wistar), and the total protein content (29.15 mg/ml) was determined using the Lowry et al's method [28]. The EROD method [29] was used to determine the activity of the cytochrome P450 isoenzyme P450-1A in the S9-fraction (51.58 pmol/ml min mg protein).…”

Section: Mutagenicity Assaysmentioning

confidence: 99%

Bacterial mutagenicity of the three isomeric dicyclopenta-fused pyrenes: the effects of dicyclopenta topology

Otero-Lobato

Jenneskens

Seinen

2004

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Cyclopenta[cd]pyrene (1) and its congeners dicyclopenta [cd,mn]-(2), dicyclopenta [cd,fg]-(3), dicyclopenta [cd,jk]pyrene (4), which were all identified as constituents of combustion exhausts, as well as their partially hydrogenated derivatives 3,4-dihydrocyclopenta[cd]-(5), 1,2,4,5-tetrahydrodicyclopenta[cd,mn]-(6), 5,6,7,8-tetrahydrodicyclopenta[cd,fg]- (7) and 1,2,6,7-tetrahydrodicyclopenta[cd,jk]pyrene (8), were assayed for mutagenicity in the Salmonella typhimurium strain TA98 using different concentrations of microsomal protein in the metabolic activation system (S9-mix, with S9-fraction from liver of Aroclor-1254-treated rats: 2, 4 and 10% (v/v), respectively). Whereas a positive mutagenic response is found for 1-4 in the presence of S9-mix, 5-8 exert no mutagenicity either with or without S9-mix. Since for 1-4 the highest response is observed with S9-mix 2% (v/v) instead of the standard 4% (v/v), a one-step activation pathway, i.e. epoxidation of the five-membered ring olefinic bonds, appears to be operational. Surprisingly, 3 and, to a lesser extent, 2 (11.7 versus 4.2 His revertants/nmol) also give a positive response in the absence of S9-mix. Hence, 2 and 3 are expected to contribute to the direct-acting mutagenicity of the non-polar fraction of combustion exhausts. Presumably for the direct-acting mutagenicity one-electron transfer processes play a role in bioactivation. The experimental observations are supported by semi-empirical AM1 calculations on the possible ultimate metabolites, i.e. mono-epoxides (2a-4a), cis-di-epoxides (2b-4b) and trans-di-epoxides (2c-4c) and the related mono-hydroxy carbocations (2d-4d and 2e-4e), and the radical anions 1 •− -4 •− .

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“…The S9-mix consists of rat-liver microsome preparations (S9-fraction) obtained from Aroclor-1254-treated male Wistar rats and NADPHgenerating co-factors. The total protein content and the activity of the cytochrome P450 isoenzyme P450-1A in the S9-fraction were determined to be 29.15 mg/ml (Lowry method) [27] and 51.58 pmol/ml/min/mg protein (EROD method) [28], respectively.…”

Section: Mutagenicity Assaysmentioning

confidence: 99%

Di-epoxides of the three isomeric dicyclopenta-fused pyrenes: ultimate mutagenic active agents

Otero-Lobato

Kaats-Richters

Havenith

et al. 2004

Mutation Research/Genetic Toxicology and Environmental Mutagene

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To rationalize the high bacterial mutagenic response recently found for the (di-) cyclopenta-fused pyrene congeners, viz. cyclopenta[cd]-(1), dicyclopenta[cd,mn]-(2), dicyclopenta[cd,fg]-(3) and dicyclopenta [cd,jk]pyrene (4), in the presence of a metabolic activation mixture (S9-mix), their (di-)epoxides at the externally fused unsaturated five-membered rings were previously proposed as the ultimate mutagenic active forms. In this study, cyclopenta[cd]pyrene-3,4-epoxide (5) and the novel dicyclopenta[cd,mn]pyrene-1,2,4,5-di-epoxide (6), dicyclopenta[cd,fg]pyrene-5,6,7,8-di-epoxide (7) and dicyclopenta[cd,jk]pyrene-1,2,6,7-di-epoxide (8) were synthesised from 1 to 4, respectively, and subsequently assayed for bacterial mutagenicity in the standard microsomal/histidine reverse mutation assay (Ames-assay with Salmonella typhimurium strain TA98). The di-epoxides 6-8 are present as a mixture of their cis-and trans-stereo-isomers in a close to 1:1 ratio ( 1 H NMR spectroscopy and ab initio IGLO/III//RHF/6-31G** calculations). The direct-acting mutagenic activity and the strong cytotoxicity exerted by 5-8 both in the absence or presence of an exogenous metabolic activation system (±S9-mix) demonstrate that the ultimate mutagenic active forms are the proposed (di-)epoxides of 1-4.

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The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers

Cited by 153 publications

References 28 publications

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

Bacterial mutagenicity of the three isomeric dicyclopenta-fused pyrenes: the effects of dicyclopenta topology

Di-epoxides of the three isomeric dicyclopenta-fused pyrenes: ultimate mutagenic active agents

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