Benzene Induces Hematotoxicity Via Acetyltransferase p300 Inhibition-Mediated Autophagy

Objective: Chronic exposure to benzene is known to be associated with hematotoxicity and is involved in the development of aplastic anemia and leukemia. However, the mechanism of benzene-induced hematotoxicity has not been well understood. Accumulating studies have reported that benzene metabolite hydroquinone (HQ) and analogous chemicals formaldehyde can induce autophagic cell death. So it is meaningful to investigate the role of autophagy and the underlying mechanisms in the benzene-induced hematotoxicity.

Materials and Methods: Human mononuclear cells were isolated from the heparinized bone marrow of healthy donors or patients with chronic benzene exposure using Ficoll gradient centrifugation, mainly consisted of progenitor and stem cells, and were measured for the levels of autophagy and the expression of p300. Totally five patients, 2 female and 3 male, all were shoe workers and had experience of contact with benzene for at least 6 months, diagnosed as occupational benzene poisoning. Five age-matched healthy donors were enrolled as controls. Besides, bone marrow mononuclear cells isolated from healthy donors were incubated with HQ 20 μM for 12 hours to determine the levels of autophagy and the expression of p300. Furthermore, thirty mice weighting 23 - 25 g were randomized into benzene group (benzene 1 ml/kg; corn oil 1 ml/kg; n=15) and corn oil group (2 ml/kg; n=15). After subcutaneous injected 12 times, three times a week, the mice executed to study the hemototoxicity of benzene with the blood cell counts, bone marrow aspiration and the pathology of the femurs. Similarly, the bone marrow mononuclear cells isolated from mice were determined for the levels of autophagy and acetylation and the expression of p300.

Results: All five enrolled patients with chronic benzene exposure had hematotoxicity. Increased autophagy, deacetylation of autophagic components and histone H3 occurred in patients with chronic benzene exposure. Furthermore, both the expression of P300 mRNA and protein were decreased significantly in patients with chronic benzene exposure compared with those in healthy donors, which arouses our interest to investigate whether in vitro exposure to benzene metabolite HQ promotes autophagy. Interesting, HQ induced cell autophagic cell death without apoptosis, which could be reversed by autophagy inhibitor 3-methyladenine and exaggerated by autophagy activator rapamycin. HQ caused deacetylation, especially decreased the acetylation of autophagy components including ATG7 and LC3. HQ also significantly decreased the acetyltransferase activity of p300 demonstrated by reduced acetylation of histone H3 and the expression of p300 mRNA and protein. HQ treatment decreased the physical interaction between p300 and autophagy components including ATG7, LC3, and ATG3. Both C646, a 'specific' antagonist of p300, and knockdown of p300 induced cell autophagy and deacetylation, similar with HQ, further identifying that p300 was a major endogenous repressor of autophagy. However, the deacetylation effect could be completely inhibited by the addition of acetyl-CoA at a concentration of 100 μM. Taken together, these data suggest that HQ promotes autophagy by inhibiting the activity of P300. We next investigate whether modulation of P300 expression regulates the autophagic flux induced by HQ. Intriguingly, Momordica Antiviral Protein 30 Kd (MAP30), a ribosome-inactivating protein isolated from dietary bitter melon, could increase p300 expression and attenuate autophagy and cell death induced by HQ. The mouse model of benzene-induced hematotoxicity was injected subcutaneously with benzene and corn oil. The levels of hemoglobin, and the numbers of white blood cells and platelets decreased significantly in benzene-treated mice compared with the vehicle mice, and the benzene-induced hematotoxicity was further verified by the bone marrow aspiration and the pathology of the femur. Similarly, the increased autophagic flux and p300 inhibition occurred in the benzene-treated mice.

Conclusion: We have demonstrated that benzene and its metabolite HQ induced autophagy by inhibiting p300, which may be a main cause of benzene-induced hematotoxicity. MAP30 can restore decreased p300 expression induced by HQ and attenuate autophagic cell death. Therefore, MAP30 may be a therapeutic drug for patients with chronic benzene exposure.