2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.

Alterations in the nigrostriatal dopamine system after acute systemic PhIP exposure

“…However, humans consume numerous HCAs (~30 known, likely many unknown) vs. PhIP alone, suggesting that considering dietary intake of a single HCA underestimates total HCA consumption (Augustsson et al , 1997; Roemer et al , 2016). Further, our previous studies found that of the two primary metabolic pathways, the N -hydroxylation product is far more neurotoxic than ring-hydroxylation (Griggs et al , 2014). N -hydroxylation is at least 13-fold less efficient in rodents vs. humans (rodent CYP1A2 converts far more PhIP to 4′-OH-PhIP than to the genotoxic and neurotoxic N -hydroxylated metabolite, N -OH-PhIP), suggesting that far higher doses may be required in rodents vs. humans to produce neurotoxic metabolites (Cheung et al , 2005; Griggs et al , 2014; Turesky et al , 1998).…”

“…Of dietary HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been extensively studied, because PhIP is the most abundant AIA isolated from the crust of cooked meat, where levels may reach ~15 micrograms/kg uncooked meat (~75% of genotoxic material) (Felton et al , 1986a; Felton et al , 1986b) While PhIP has been extensively studied as a mutagen, effects on the nervous system have not yet been substantially evaluated. Recently, our group showed that PhIP is selectively toxic to dopaminergic neurons in primary rat midbrain cultures (Griggs et al , 2014). In that study, PhIP and its bioactive phase I metabolite, N -OH-PhIP induced loss of dopaminergic neurons and neurite retraction, whereas nondopaminergic neurons were spared.…”

Dietary Factors in the Etiology of Parkinson’s Disease

“…K. Turteltaub and A. Director-Myska). Recent neurotoxicity studies on PhIP in vitro using primary midbrain cultures have been performed [ 192 ]. PhIP and N-OH-PhIP treatments at 1 μ M were selectively toxic to primary dopaminergic neurons isolated from embryonic day 17 rat embryos.…”

Analysis of Statistical Methods Currently used in Toxicology Journals

“…There are some outliers which strongly influenced the mean, with sample size being 71, 184, and 694. The paper which employed large sample sizes of 184 and 694 were about neurotoxicity of heterocyclic amines but the definition of sample number were not conventional (number of neurites for the outcome measurement of neurite length, and mean pixel intensity in the regions of interests) (32) while the one used the average sample size of 71 were about the neurotoxicity of flame retardants and the sample number is the number of wells (of 96 well plate) per group (22) . Besides these few outliers, most studies have small sample sizes, raising a concern that the studies may be underpowered to conduct inferential statistics and the dispersion of data might not indicate the true distribution of the underlying distribution of the outcome variables.…”