Comparison of metabolite profiles generated in Aroclor‐induced rat liver and human liver subcellular fractions: Considerations for in vitro genotoxicity hazard assessment

Abstract

Because it is well known that metabolites of chemicals and drugs are frequently the ultimate species responsible for genotoxicity and carcinogenicity, in vitro testing to identify the human genotoxicity hazard potential of new chemicals and drugs routinely utilizes liver S-9 fraction from rats treated with Aroclor 1254 as a system that can generate metabolites. However, it is frequently questioned as to whether such an in vitro metabolite generation system is the most relevant for human risk, or whether the assay would be better served by using a human-derived in vitro system. To address this, 16 common drugs have been examined for profiles of metabolites in Aroclor-induced rat liver S-9 and pooled human liver S-9. Metabolite profiles were compared using high pressure liquid chromatography coupled with ion trap mass spectrometry, in line with ultraviolet or radiometric detection to help make semiquantitative comparisons. Results showed that, with few exceptions, metabolites generated in the human system were also generated in the rat system. Also, in several cases the rat system generated considerably more metabolites, suggesting that there is a potential that positive genotoxicity findings could be caused by metabolites that have no relevance to humans. These findings suggest that when conducting in vitro genotoxicity testing using the Aroclor-induced rat liver S-9 system, knowledge of the metabolite profile in the system is important, and a comparison to the profile generated in human liver S-9 could be of value when interpreting the genotoxicity results.

In Vitro Recapitulating of TP53 Mutagenesis in Hepatocellular Carcinoma Associated With Dietary Aflatoxin B1 Exposure

“…We had also verified that the herein used rat liver S9-activation system, similarly to human liver S9 preparations or the classical double-induced S9 mix (Aroclor + phenobarbital or dexamethasone), has the capacity to convert AFB 1 to DNA reactive derivatives in vitro 29, 30. The rat liver S9-activation system is proven to contain cytochrome P450 3A4 and 1A2 enzymes 38 that are primarily involved in oxidative bioactivation of AFB 1 12–14. Thus, we included the S9-activation system in our cytotoxicity experiments and established a relevant dosing range for AFB 1 , which served as a guideline for DNA adduct and mutagenesis experiments ( see , Fig.…”

Application of a New Genotoxicity Test System with Human Hepatocyte Cell Lines to Improve the Risk Assessment in the Drug Development

“…Some phase II enzymes, such as UDPglucuronosyltransferases (UGT), glutathione S-transferases (GST), sulfotransferase (SULT), or N-acetyl transferases, are not active in the reduced form of the nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented S9 system (S9 mix) because other cofactors and additives (e.g., uridine diphosphate glucuronic acid, glutathione, acetylcoenzyme A, etc.) would be needed (Ku et al, 2007;Obach and Dobo, 2008). This can be essential not only for reducing potential false positives (e.g., reactive electrophiles that would be rapidly quenched by conjugation in vivo before being able to cause mutation) but also for false negatives because some conjugation reactions can yield metabolites that are more reactive than their substrate (e.g., sulfation of N-hydroxy-2-acetylaminofluorene or acetylation of N-hydroxylated heterocyclic amines) (Dashwood, 2002;Ku et al, 2007).…”

“…This is because the system is set up to favor CYP-mediated metabolism and the other enzymes present in the system that could be responsible for detoxication of reactive intermediates are not supplemented with the appropriate cofactors (e.g., UGT, GST, methyl transferases, etc), thus potentially providing an unrealistic metabolic profile. In a recent work by Obach and Dobo, it was revealed that many human in vivo metabolites arise via conjugation reactions with the limited 16 drugs (Obach and Dobo, 2008). This can be important not only for reducing potential false positives (e.g., reactive electrophiles that would be rapidly quenched by conjugation in vivo before being able to cause mutation) but can also be important for false negatives because some conjugation reactions can yield metabolites that are more reactive than their substrate (e.g., sulfation of aliphatic alcohols or glucuronidation of carboxylic acids; [Glatt et al, 1998;Sallustio et al, 2006]).…”

“…However are these are also not active in the standard testing system because the necessary cofactor, 3'-phosphoadenosine-5'-phosphosulfate, is not added (Glatt, 2000;Glatt, 2005). From the study by Obach and Dobo using 16 drugs commonly used, not all metabolites observed as significant in humans in vivo are generated in the system using rat or human S9 fractions (Obach and Dobo, 2008).. Furthermore, they reported that a metabolite observed in humans in vivo was only seen in the rat S9 system and not the human S9 system in a few cases, (Obach and Dobo, 2008).…”

“…From the study by Obach and Dobo using 16 drugs commonly used, not all metabolites observed as significant in humans in vivo are generated in the system using rat or human S9 fractions (Obach and Dobo, 2008).. Furthermore, they reported that a metabolite observed in humans in vivo was only seen in the rat S9 system and not the human S9 system in a few cases, (Obach and Dobo, 2008). Many human in vivo metabolites arise via conjugation reactions, which will not be observed in the in vitro S9 system as presently supplemented in standard in vitro genotoxicity tests.…”