N-Hydroxylation of 4-Aminobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse Model of Chemically Induced Liver Cancer

Wang, Shuang; Sugamori, Kim S.; Tung, A. K.; McPherson, J. Peter; Grant, Denis M.

doi:10.1093/toxsci/kfv006

Cited by 15 publications

(19 citation statements)

References 42 publications

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“…[18][19][20] Several CYP2E1 dependent mechanisms contributing to tumorigenesis have been suggested including formation of toxic intermediate derivatives and activated carcinogens. [21][22][23] CYP2E1 mediated ROS generation could also contribute to tumor development through pathways in which ROS play vital role such as DNA damage, enhanced angiogenic responses [24] autophagy [4,25,26] ER stress [27] and unfolded protein response (UPR). [28] Furthermore, research in our laboratory has indicated that CYP2E1 is differentially expressed in a manner dependent on the genetic background and the stage of breast cancer, regulating oxidative stress response and metastasis.…”

Section: Introductionmentioning

confidence: 99%

Oxidative stress and breast cancer biomarkers: the case of the cytochrome P450 2E1

Singh

Rajendran

Kuroda

et al. 2016

JCMT

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Aim:The aim of the study is to investigate the impact of the cytochrome P450 2E1, which is the most efficient CYP450 family member in generating reactive oxygen species (ROS), on cellular energy metabolism of breast cancer cells and therefore the effects of CYP2E1 on breast carcinogenesis. Methods: The estrogen receptor positive MCF-7 and the triple negative MDA-MB-231 breast cancer cells were used as experimental system to estimate ROS generation in these cells overexpressing CYP2E1 and treated with the glycolytic inhibitors 3-bromopyruvate or 2-deoxyglucose in the presence or absence of the CYP2E1 inhibitor chlormethiazole. Adenosine triphosphate (ATP) assay was used to measure ATP production and lactate assay to quantify the efflux of lactic acid in breast cancer cells treated with the CYP2E1 inhibitor chlormethiazole, the mitochondrial membrane potential and cell viability assays were employed to assess the pathway of cellular energy production and cellular death respectively after treatment of MCF-7 and MDA-MB-231 with the CYP2E1 activator acetaminophen or the CYP2E1 inhibitor chlormethiazole. Results:The results indicated increased ROS generation in breast cancer cells overexpressing CYP2E1. ROS generation was differentially regulated in breast cancer cells upon treatment with the CYP2E1 inhibitor chlormethiazole. Chlormethiazole treated MCF-7 cells exhibited reduced lactate efflux implying that CYP2E1 directly or indirectly regulates the glycolytic rate in these cells. Furthermore the mitochondrial membrane potential of both MCF-7 and MDA-MB-231 cells was differentially affected by the CYP2E1 activator acetaminophen versus the CYP2E1 inhibitor chlormethiazole providing additional support for the involvement of CYP2E1 in energy metabolic pathways in breast cancer. Conclusion: Results presented in this study provide evidence to suggest that CYP2E1 regulates cellular energy metabolism of breast cancer cells in a manner dependent on cell type and potentially on the clinical staging of the disease therefore CYP2E1 is a possible breast cancer biomarker.

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Section: Introductionmentioning

confidence: 99%

Oxidative stress and breast cancer biomarkers: the case of the cytochrome P450 2E1

Singh

Rajendran

Kuroda

et al. 2016

JCMT

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“…Isolation of genomic DNA from mouse tail clips was carried out as described previously (Sugamori et al, 2003). Genotyping for Cyp2e1(2/2) mice was carried out as described previously (Wang et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

“…Liver microsomes were prepared from 7-to 8-week-old mice as described previously (Wang et al., 2015). In brief, livers were homogenized, centrifuged at 9000 Â g for 20 minutes, and the supernatant was further centrifuged at 105,000 Â g for 60 minutes to pellet the microsomal fraction.…”

Section: Methodsmentioning

confidence: 99%

“…Cell culture and transient transfection conditions for the Hepa1c1c7 mouse hepatoma cell line were carried out as described previously (Wang et al, 2015). In brief, cells were seeded at 5 Â 10 5 cells per well onto six-well plates, grown overnight, and treated with transfection mix (6 ml of Lipofectamine LTX:2 mg of DNA for CYP1A2 and 9 ml Lipofectamine LTX:2 mg of DNA for CYP2E1) for 6 hours in minimum essential medium (Life Technologies Inc.) only, followed by a change back to complete growth medium at 6 hours.…”

Section: P450-dependent Metabolism Of 4-aminobiphenyl In Mice 917mentioning

confidence: 99%

“…We recently identified CYP2E1 as a novel and significant ABP N-hydroxylating enzyme in liver microsomes from postnatal day 15 mice-the age at which exposure to ABP occurs in the standard neonatal tumor bioassay (Wang et al, 2015). In that study, Cyp2e1(2/2) neonates had ;50% lower ABP N-hydroxylation activity than wild-type mice.…”

Section: Introductionmentioning

confidence: 97%

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Relative Contributions of CYP1A2 and CYP2E1 to the Bioactivation and Clearance of 4-Aminobiphenyl in Adult Mice

et al. 2015

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4-Aminobiphenyl (ABP), a prototypical aromatic amine carcinogen in rodents and humans, requires bioactivation to manifest its toxic effects. A traditional model of ABP bioactivation, based on in vitro enzyme kinetic evidence, had postulated initial N-hydroxylation by the cytochrome P450 isoform CYP1A2. This is followed by phase 2 O-conjugation and hydrolysis to form a reactive nitrenium ion that covalently binds to DNA and produces tumor-initiating mutations. However, Cyp1a2(2/2) mice still possess significant liver ABP N-hydroxylation activity, DNA damage, and incidence of ABP-induced liver tumors, and in vivo induction of CYP1A2 paradoxically reduces levels of ABP-induced DNA damage. Competing ABP detoxification pathways can include N-acetylation by arylamine N-acetyltransferase 1 (NAT1) and/or NAT2; however, wild-type and Nat1/2(2/2) mice have similar in vivo ABP clearance rates. Together, these studies suggest the existence of novel ABP bioactivating and clearance/detoxification enzymes. In the present study, we detected similar reductions in V max for ABP N-hydroxylation by liver microsomes from Cyp1a2(2/2) and Cyp2e1(2/2) mice when compared with wild-type mice. In addition, recombinant mouse CYP1A2 and CYP2E1 were both able to N-hydroxylate ABP in mouse hepatoma cells. However, the in vivo clearance of ABP was significantly reduced in Cyp1a2(2/2) but not in Cyp2e1(2/2) mice. Our results support a significant role for CYP2E1 as a novel ABP N-oxidizing enzyme in adult mice, and suggest a more important contribution of CYP1A2 to the in vivo plasma clearance and thus detoxification of ABP.

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Mutagenicity of N‐hydroxy‐4‐aminobiphenyl in human TP53 knock‐in (Hupki) mouse embryo fibroblasts

Hölzl-Armstrong

Kucab

Zwart

et al. 2021

Environ and Mol Mutagen

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TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knockin mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 μM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (40 adducts/10 8 nucleotides) was detected after 24 hr treatment with 40 μM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.

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N-Hydroxylation of 4-Aminobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse Model of Chemically Induced Liver Cancer

Cited by 15 publications

References 42 publications

Oxidative stress and breast cancer biomarkers: the case of the cytochrome P450 2E1

Oxidative stress and breast cancer biomarkers: the case of the cytochrome P450 2E1

Relative Contributions of CYP1A2 and CYP2E1 to the Bioactivation and Clearance of 4-Aminobiphenyl in Adult Mice

Mutagenicity of N‐hydroxy‐4‐aminobiphenyl in human TP53 knock‐in (Hupki) mouse embryo fibroblasts

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