Parkinson's disease (PD) affects nearly one million people in the United States. Dopamine depletion due to the loss of dopaminergic neurons is a hallmark of PD. The β-carboline harmane, a neurotoxic heterocyclic aromatic amine (HAA), crosses the blood-brain barrier and its levels are elevated in cerebrospinal fluid of PD patients. The direct brain infusion with other HAAs, including 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, and norharman depletes dopamine, suggesting a possible role for other HAAs in PD etiology. We have extended studies to the aminoimidazoarene class of HAAs, which prominently form in cooked meats. We identified 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as being selectively toxic to dopaminergic neurons. PhIP is a possible human carcinogen abundantly formed in cooked red meat. In humans, PhIP undergoes metabolism to form the genotoxic N-hydroxylated intermediate (HONH-PhIP). Conjugation enzymes, including N-acetyltransferases (NATs) and sulfotransferases (SULTs), convert HONH-PhIP to reactive conjugates that covalently adduct to DNA. Employing SH-SY5Y, a human neuroblastoma cell line, as a model for PD-related neurotoxicity, we examined the cytotoxicity and DNA damage of PhIP, other HAAs, including 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline, and 2-amino-9H-pyrido[2,3-b]indole, and their synthesized HONH-HAA metabolites. The parent amines are poorly bioactivated in SY5Y cells. However, HONH-PhIP induced a dose-dependent cytotoxicity and underwent extensive bioactivation, leading to the formation of high DNA adduct levels compared to other HONH-HAAs. PhIP-DNA adducts were significantly higher and more persistent in mitochondrial DNA (mtDNA) than nuclear DNA (nDNA). PhIP DNA adducts formed in a dose-dependent manner in both mtDNA and nDNA at doses approaching environmental exposure levels. Notably, mtDNA damage is a major mechanism in developing PD. DNA binding assays with cytosolic fractions of SH-SY5Y fortified with AcCoA, PAPS, or ATP revealed that AcCoA dependent enzymes mainly carried out bioactivation of HONH-PhIP. DNA binding assays with mitochondrial fraction fortified with AcCoA, PAPS, and ATP are under study. Specific inhibitors will be employed to determine the roles of cytoplasmic choline acetyltransferase and the mitochondrial carnitine acetyltransferase in HONH-PhIP bioactivation in SH-SY5Y cells. These data provide support for a possible role of PhIP in PD etiology.

Mitochondrial and nuclear DNA damage by the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in human neuroblastoma cell line

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