Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b5, which can also act as an electron donor from cytochrome b5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP–DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP–DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2162-7) contains supplementary material, which is available to authorized users.
Cytochrome P450 enzyme systems have been widely used in vitro to determine the pathways of activation of procarcinogens, but paradoxically, these same enzymes can play a more predominant role in carcinogen detoxification in vivo.
Many studies have shown that algal growth is enhanced by organic carbon and algal mixotrophy is relevant for physiology and commercial cultivation. Most studies have tested only a single organic carbon concentration and report different growth parameters which hampers comparisons and improvements to algal cultivation methodology. This study compared growth of green algae Chlorella vulgaris and Chlamydomonas reinhardtii across a gradient of photoautotrophic-mixotrophic-heterotrophic culture conditions, with five acetate concentrations. Culture growth rates and biomass achieved were compared using different methods of biomass estimation. Both species grew faster and produced the most biomass when supplied with moderate acetate concentrations (1–4 g L−1), but light was required to optimize growth rates, biomass yield, cell size and cell chlorophyll content. Higher acetate concentration (10 g L−1) inhibited algal production. The choice of growth parameter and method to estimate biomass (optical density (OD), chlorophyll a fluorescence, flow cytometry, cell counts) affected apparent responses to organic carbon, but use of OD at 600, 680 or 750 nm was consistent. There were apparent trade-offs among exponential growth rate, maximum biomass, and culture time spent in exponential phase. Different cell responses over 1–10 g L−1 acetate highlight profound physiological acclimation across a gradient of mixotrophy. In both species, cell size vs cell chlorophyll relationships were more constrained in photoautotrophic and heterotrophic cultures, but under mixotrophy, and outside exponential growth phase, these relationships were more variable. This study provides insights into algal physiological responses to mixotrophy but also has practical implications for choosing parameters for monitoring commercial algal cultivation.
The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.
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