A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. A polymorphism in NQO1, a C609T substitution, has been identified, and individuals homozygous for this change (T/T) have no detectable NQO1. Exposed workers with a T/T genotype have an increased risk of benzene hematotoxicity. This finding suggests NQO1 is protective against benzene toxicity, which is difficult to reconcile with the lack of detectable NQO1 in human bone marrow. The human promyeloblastic cell line, KG-1a, was used to investigate the ability of the benzene metabolite hydroquinone (HQ) to induce NQO1. A concentration-dependent induction of NQO1 protein and activity was observed in KG-1a cells cultured with HQ. Multiple detoxification systems, including NQO1 and glutathione protect against benzene metabolite-induced toxicity. Indeed, exposure to a noncytotoxic concentration of HQ induced both NQO1 and soluble thiols and protected against HQ-induced apoptosis. NQO1 protein and activity increased in wild-type human bone marrow cells (C/C) exposed to HQ, whereas no NQO1 was induced by HQ in bone marrow cells with the T/T genotype. Intermediate induction of NQO1 by HQ was observed in heterozygous bone marrow cells (C/T). NQO1 also was induced by HQ in wild-type (C/C) human bone marrow CD34(+) progenitor cells. Our data suggest that failure to induce functional NQO1 may contribute to the increased risk of benzene poisoning in individuals homozygous for the NQO1 C609T substitution (T/T).

Figure 1

NQO1 induction in KG-1a cells exposed to benzene metabolites. (A) NQO1 activity was measured after treatment with 0–100 μM HQ (black bars) or 0–100 μM CAT (gray bars) for 24 h. Activities are reported as nmol 2,6-dichlorophenol-indophenol reduced/min per mg cellular protein. Data are presented as mean ± SD (n = 3). ∗, Significantly greater than control (P ≤ 0.05). (B) NQO1 protein was determined by immunoblot analysis after above treatments. Purified recombinant human NQO1 protein (5 ng) was included as a standard (NQO1). Immunoblots are representative of three separate experiments for each compound.

Figure 2

Pretreatment with HQ increased soluble thiols in KG-1a cells. KG-1a cells were cultured with 10 μM HQ or vehicle control (PBS) for 24 h. Data are presented as mean nmol thiol/mg protein ± SD (n = 5). ∗, Significantly greater than control (P ≤ 0.05).

Figure 3

Protection against HQ-induced apoptosis in KG-1a cells after induction of NQO1. Cells were pretreated with 10 μM HQ (○) or vehicle control (PBS, ●) for 24 h. Subsequently, cells were treated with HQ (0–200 μM) for 12 h after which apoptosis was measured. (A) Apoptosis was determined based on morphological criteria. Data are presented as mean percentage apoptotic cells ± SD (n = 3). ^†, Percentage of apoptosis is significantly greater than control (P ≤ 0.05). ∗, Protection is significant (P ≤ 0.05). (B) Chromatin degradation into large fragments was measured by field inversion gel electrophoresis. Gel is representative of three separate experiments.

Figure 4

Pretreatment with HQ failed to protect against apoptosis induced by compounds other than HQ. KG-1a cells were pretreated with 10 μM HQ or vehicle control for 24 h. Subsequently, cells were exposed to various inducers of apoptosis for 12 h. Data are presented as mean percentage apoptotic cells ± SD (n = 4). ∗, Significantly different from that observed with the corresponding treatment in cells pretreated with PBS (P ≤ 0.05).

Figure 5

Effect of the C609T substitution on induction of NQO1 by HQ in human bone marrow cells. (A) NQO1 activity was measured after bone marrow cells C/C (●), C/T (■), or T/T (▴) at position 609 in the coding region of NQO1 were treated with 0–100 μM HQ for 24 h. Activities are reported as nmol 2,6-dichlorophenol-indophenol reduced/min per mg cellular protein. Data are presented as mean ± SD (n ≥ 3). ∗, Significantly greater than control (P ≤ 0.05). (B) NQO1 protein was measured by immunoblot analysis. Representative immunoblots of NQO1 protein in HQ-treated cells with the C/C, C/T, or T/T genotype are shown (n ≥ 3 for each genotype). Purified recombinant human NQO1 protein (5 ng) was included as a standard (NQO1). Freshly isolated bone marrow cells (F) lacked detectable NQO1 protein in all cases.

Figure 6

Induction of NQO1 in C/C bone marrow cells after exposure to 0–100 μM CAT. (A) NQO1 activity was measured 24 h after treatment and is reported as nmol 2,6-dichlorophenol-indophenol reduced/min per mg cellular protein. Data are represented as mean ± SD (n = 3). ∗, NQO1 activity was significantly greater than control (P ≤ 0.05). (B) NQO1 protein was measured by immunoblot analysis. Purified recombinant human NQO1 was included as a standard (5 ng, NQO1). No NQO1 protein was detected in freshly isolated bone marrow cells (F). Immunoblot is representative of three separate experiments.

Figure 7

Induction of NQO1 in human bone marrow mononuclear cells enriched for CD34 expression. Isolated CD34⁺ cells were cultured with or without HQ (10 μM) for 18 h. All three donors were genotyped as C/C at position 609 of the coding region of NQO1. (A) NQO1 activity. Data are presented as mean ± SD (n = 3). ∗, Significantly greater than control (P ≤ 0.05). (B) NQO1 protein was measured by immunoblot analysis. Purified recombinant human NQO1 was included as a standard (0.5 ng, NQO1). Immunoblot is representative of three separate experiments. No NQO1 activity or protein was detected in freshly isolated CD34⁺ cells (F).