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1Department of Toxicology, School of Public Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, Southern Medical University, Guangzhou, China.
Abstract:
The accessibility of reactive metabolites to test cells is critical for a genotoxic response. However, sulfo-conjugates formed outside may not readily enter cells, and some metabolites formed by cytochromes P450 (CYPs) may not endure transport. This topic was addressed in the present study, using V79 cells engineered for human CYPs and/or a sulfotransferase (SULT). First, 1-methylpyrene, 1-hydroxymethylpyrene, benzo[a]pyrene, and aflatoxin B1 significantly induced micronuclei in V79-hCYP1A2-hSULT1A1, V79-hSULT1A1, V79-hCYP1A1, and V79-hCYP1A2 cells, respectively. Subsequently, we used these cell lines as external activating systems in various experimental settings in combination with V79-derived target cells lacking critical enzymes. 1-Methylpyrene (activated by CYPs and SULTs sequentially) showed an activity similar to that in V79-hCYP1A2-hSULT1A1 cells, in each following model: a mixed V79-hCYP1A2:V79-hSULT1A1 (1:1) culture, exposure of V79-hCYP1A2 to 1-methylpyrene followed by transfer of medium to V79-hSULT1A1 target cells, and V79-hSULT1A1 communicating with V79-hCYP1A2 through 0.4-μm pores and over a 1-mm distance in a unique transwell system. These results suggest ready transfer of 1-hydroxymethylpyrene formed in V79-hCYP1A2 to V79-hSULT1A1 for further activation. In the last two models, with V79-hSULT1A1 for activation and V79-Mz as target, 1-hydroxymethylpyrene induced micronuclei mildly, suggesting limited intercellular transfer of the ultimate genotoxicant, 1-sulfooxymethylpyrene. Benzo[a]pyrene induced micronuclei in V79-Mz communicating with V79-hCYP1A1 via porous membranes, whereas aflatoxin B1 was inactive in V79-Mz communicating with V79-hCYP1A2. Our results suggest that the sulfo-conjugate tested may have difficulty entering cells for a genotoxic effect, and the reactive metabolite of aflatoxin B1, unlike that of benzo[a]pyrene, could not travel an adequate distance to enter cells. Environ. Mol. Mutagen. 61:224-234, 2020. © 2019 Wiley Periodicals, Inc.
Insights
Reactive metabolites must enter cells to cause genotoxicity. This study shows sulfo-conjugates may struggle to enter cells, limiting their genotoxic response, unlike other reactive metabolites.
Area of Science:
- Toxicology
- Genetics
- Molecular Biology
Background:
- Reactive metabolites are crucial for genotoxic responses.
- The accessibility of these metabolites to target cells is a key factor.
- Metabolites formed extracellularly or by cytochromes P450 (CYPs) may face transport challenges.
Purpose of the Study:
- To investigate the role of cell accessibility in genotoxic responses.
- To determine if sulfo-conjugates, formed extracellularly, can readily enter cells.
- To compare the intercellular transport and genotoxicity of metabolites from different procarcinogens.
Main Methods:
- Utilized V79 cells engineered with human CYPs and/or sulfotransferases (SULTs).
- Assessed genotoxicity via micronucleus induction in engineered cell lines.
- Employed various experimental settings, including co-cultures and transwell systems, to study intercellular metabolite transfer.
Main Results:
- 1-Methylpyrene, benzo[a]pyrene, and aflatoxin B1 induced micronuclei in engineered V79 cells.
- Sequential activation of 1-methylpyrene by CYPs and SULTs showed significant genotoxicity.
- Limited intercellular transfer of the ultimate genotoxicant 1-sulfooxymethylpyrene was observed, suggesting poor cell entry.
- Benzo[a]pyrene metabolites entered target cells, while aflatoxin B1 metabolites did not travel adequately.
Conclusions:
- The ability of reactive metabolites to enter target cells significantly impacts genotoxic outcomes.
- Sulfo-conjugates may exhibit reduced genotoxicity due to difficulties in cellular uptake.
- Intercellular transport distances and cell entry mechanisms vary for different procarcinogen metabolites.
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