Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke

Abstract

Background: Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1.

Methods: Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed.

Results: Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia.

Conclusions: Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings.

Figure 1

The Ras status of yeast cells modulates the toxicity of HQ. The Area Under Curve (AUC) was calculated for each strain after 24 h of exposure to the indicated doses of HQ. The bars represent mean AUC as a percentage of the untreated for each strain with standard error of three replicates. Sensitivity was determined by comparison to the wild type strain (gray bars = wild type; white bars = indicated deletion/overexpression strain). Aira2Δ (increased Ras signaling) is sensitive to HQ. Bras1Δ (reduced Ras signaling) is resistant to HQ. Cras2Δ showed a response to HQ equivalent to WT. D Overexpression of RAS1 (increased Ras signaling) results in sensitivity to HQ.

Figure 2

Detection of genotoxicity through in vitro erythropoiesis. A Representative micrograph of Day 2 harvested cultures at 100x magnification. The arrows indicate normally enucleated PCEs, and the arrowhead indicates a micronucleated PCE. Scale bar = 8 μM. BNf1^−/− Lin- BM cultures exhibit sensitivity to MN formation after in vitro exposure to HQ. The response of this erythropoietic culture system to HQ treatment is quantified by MN frequency in PCEs. Data presented is the mean of the “n” independent cultures ± SD.

Figure 3

Nf1^−/− hematopoietic progenitors of the granulocyte and macrophage lineages are resistant to HQ-induced toxicity. CFU-GM colony counts, relative to untreated controls, are plotted vs. HQ exposure level for cultures derived from both WT and Nf1^−/− BM. Bone marrow cells from each genotype were treated with HQ in MethoCult® capable of inducing growth of CFU-GM colonies and then cultured for 12 days. In both genotypes colony formation was reduced upon exposure to as little as 10 μM HQ. CFU-GM numbers continued to fall with increasing HQ exposure up to 30 μM HQ treatment, and at all doses the Nf1^−/− BM appeared resistant to HQ-induced toxicity when compared with BM from WT littermates.