Comparative Dopaminergic Neurotoxicity of Heterocyclic Amines in Primary Midbrain Cultures.

Angela A Cruz-Hernandez, Purdue University

Abstract

There are many different factors that contribute to the etiology of Parkinsons Disease (PD). The exact etiology is unknown, but dietary factors are one emerging factor of interest in the etiology. Heterocyclic amines (HCAs) have been implicated in cancers and tumors due to their mutagenic properties. There are currently >20 known HCAs that are produced by different cooking methodologies. HCAs are primarily formed in meats cooked at high temperatures. Recent data identified HCA-induced neurotoxicity. Previous findings in our laboratory have indicated, 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine (PhIP) as selectively neurotoxic to dopaminergic (DA) neurons in rat primary midbrain neuronal cell culture. The aim of this study is to determine the toxicity of additional prominent dietary HCAs that are found in meat at different concentrations. We tested the following dietary HCAs, representing multiple HCA subclasses (imidazoazaarenes and α/β-carbolines): 3-methyl-3H-imidazo[4,5-f]quinolin-2-amine (IQ), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2- Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinolone (MeIQ), 3,4,8-trimethylimidazo[4,5-f]quinoxalin-2-amine (4-8 DiMeIQx), and 2-Amino-9H-pyrido[2,3-b]indole (AαC). Primary midbrain neuronal cultures were prepared using E17 rat embryos and stained for tyrosine hydroxylase (TH, marker for DA neurons) and microtubule-associated protein (MAP2, pan-neuronal). This model was used to test DA neurotoxicity at a range of HCA doses (100nM - 5μM). Viability was assessed by counting MAP2- and TH-immunoreactive primary neurons in a blinded manner. One-way ANOVA with Dunnetts post-hoc test was used for statistical analysis. A statistically significant decrease in the percentage of DA neurons emerged in all compounds at different concentrations. The most toxic compounds, which had an effect at all doses from 100nM-5 μM, were 4,8-DiMeIQx and Norharman. TH neurite length confirmed HCAs are selectively toxic to DA neurons. HCAs did not exhibit an effect on the non-DA neurons through the quantification of MAP2+ cell counts and neurite length. Thus, our data shows that multiple HCAs produce selective DA neuron toxicity in primary midbrain culture and that compounds from multiple HCA subclasses were able to produce such neurotoxicity. Mechanistic studies are underway to determine how structural differences influence mechanisms of HCA-induced neurotoxicity and to further determine how HCA exposure may be important in PD.

Degree

M.S.

Advisors

Cannon, Purdue University.

Subject Area

Toxicology

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