Cashmeran as a potential endocrine disrupting chemical: What does the weight-of-the-evidence indicate?

Cashmeran is a fragrance ingredient. Risk assessments are available but have not focused on its endocrine disruptor potential. The objective was to evaluate Cashmeran as a potential endocrine-disrupting chemical (EDC). The assessment was based on data from US EPA's CompTox Chemicals Dashboard, the Danish (Q)SAR Database, in vitro assays, and in vivo studies. ToxCast assays related to estrogen, androgen, thyroid, and steroidogenesis modalities were Inactive at non-cytotoxic concentrations. In vitro assays demonstrated no estrogenic activity in a human cervical epithelioid carcinoma HeLa cell line and indicated only weak agonist estrogenic activity in Chinese Hamster Ovary (CHO)-K1 cells. In the same test, no agonist or antagonist activity was detected for human androgen receptor (hAR) and thyroid hormone receptor ß (hTHRß) binding. The Danish QSAR database didn't indicate any ED potential. There were no adverse endocrine related effects in either a 90-day repeated gavage dosing study or a reproductive and developmental screening study. Regarding ED potential for environment, the data from two limited environmental ED related studies on Cashmeran did not raise any concern. Data from in vitro and in vivo studies were considered for environmental ED concern. Based on the weight-of-the-evidence, Cashmeran is not expected to cause endocrine effects.

Hypoxia diminishes the detoxification of the environmental mutagen benzo[a]pyrene.

Hypoxia promotes genetic instability and is therefore an important factor in carcinogenesis. We have previously shown that activation of the hypoxia responsive transcription factor HIFα can enhance the mutagenic phenotype induced by the environmental mutagen benzo[a]pyrene (BaP). To further elucidate the mechanism behind the ability of hypoxia to increase mutagenicity of carcinogens, we examined the activation and detoxification of BaP under hypoxic conditions. To this end, the human lung carcinoma cell line A549 was treated with BaP under 20%, 5% or 0.2% oxygen for 18h and alterations in BaP metabolism were assayed. First, BaP-induced expression of key metabolic enzymes was analysed; expression levels of the activating CYP1A1 and CYP1B1 were increased, while the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. To evaluate whether these changes had an effect on metabolism, levels of BaP and several of its metabolites were determined. Cells under hypoxia have a reduced capacity to metabolise BaP leaving more of the parent molecule intact. Additionally, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations. Finally, under hypoxia, DNA adducts accumulated over a period of 168 h, whereas adducts were efficiently removed in 20% oxygen conditions. The delayed detoxification kinetics resulted in a 1.5-fold increase in DNA adducts. These data indicate that the metabolism under hypoxic conditions has shifted towards increased activation of BaP instead of detoxification and support the idea that modulation of carcinogen metabolism is an important additional mechanism for the observed HIF1 mediated genetic instability.

Loss of VHL in RCC Reduces Repair and Alters Cellular Response to Benzo[a]pyrene.

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene occur in the majority of sporadic renal-cell carcinomas (RCC). Loss of VHL function is associated with stabilization of hypoxia-inducible factor α (HIFα). We and others demonstrated that there is a two-way interaction between the aryl hydrocarbon receptor, which is an important mediator in the metabolic activation and detoxification of carcinogens, and the HIF1-pathway leading to an increased genetic instability when both pathways are simultaneously activated. The aim of this study was to investigate how environmental carcinogens, such as benzo[a]pyrene (BaP), which can be metabolically activated to BaP-7,8-diOH-9,10-epoxide (BPDE) play a role in the etiology of RCC. We exposed VHL-deficient RCC4 cells, in which HIFα is stabilized regardless of oxygen tension, to 0.1 µM BaP for 18 h. The mutagenic BPDE-DNA adduct levels were increased in HIFα stabilized cells. Using qRT-PCR, we demonstrated that absence of VHL significantly induced the mRNA levels of AhR downstream target CYP1A1. Furthermore, HPLC analysis indicated that loss of VHL increased the concentration of BaP-7,8-dihydroxydiol, the pre-cursor metabolite of BPDE. Interestingly, the capacity to repair BPDE-DNA adducts in the HIFα stabilized RCC4 cells, was markedly reduced. Taken together, these data indicate that loss of VHL affects BaP-mediated genotoxic responses in RCC and decreases repair capacity.

Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by benzo(a)pyrene ex vivo.

Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype-phenotype relationships and gene-gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase (CAT, rs1001179) and cytochrome P450 1B1 (CYP1B1, rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 (R = 0.92, P < 0.001). To further investigate the mechanism by which catalase influences CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 (P < 0.05). We conclude that the genetic polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo.

Decreased nucleotide excision repair in steatotic livers associates with myeloperoxidase-immunoreactivity.

Chronic inflammation is characterized by the influx of neutrophils and is associated with an increased production of reactive oxygen species that can damage DNA. Oxidative DNA damage is generally thought to be involved in the increased risk of cancer in inflamed tissues. We previously demonstrated that activated neutrophil mediated oxidative stress results in a reduction in nucleotide excision repair (NER) capacity, which could further enhance mutagenesis. Inflammation and oxidative stress are critical factors in the progression of nonalcoholic fatty liver disease that is linked with enhanced liver cancer risk. In this report, we therefore evaluated the role of neutrophils and the associated oxidative stress in damage recognition and DNA repair in steatotic livers of 35 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n=17) or steatosis alone (n=18). The neutrophilic influx in liver was assessed by myeloperoxidase (MPO) staining and the amount of oxidative DNA damage by measuring M(1)dG adducts. No differences in M(1)dG adduct levels were observed between patients with or without NASH and also not between individuals with high or low MPO immunoreactivity. However, we found that high expression of MPO in the liver, irrespective of disease status, reduced the damage recognition capacity as determined by staining for histone 2AX phosphorylation (γH2AX). This reduction in γH2AX formation in individuals with high MPO immunoreactivity was paralleled by a significant decrease in NER capacity as assessed by a functional repair assay, and was not related to cell proliferation. Thus, the observed reduction in NER capacity upon hepatic inflammation is associated with and may be a consequence of reduced damage recognition. These findings suggest a novel mechanism of liver cancer development in patients with nonalcoholic fatty liver disease.

Diminished carcinogen detoxification is a novel mechanism for hypoxia-inducible factor 1-mediated genetic instability.

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1alpha activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl(2), which leads to HIF-1alpha stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and gamma-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.