Original ContributionMethamphetamine-enhanced embryonic oxidative DNA damage and neurodevelopmental deficits☆
Introduction
Methamphetamine (METH), commonly known as “speed,” “crystal,” or “ice,” is a potent and addictive psychomotor stimulant taken by an estimated 8.8 million people (4.0% of the population) in the United States at some time in their lives [1].
Although METH is known to be a potentially neurotoxic agent to adult animal models and humans [2], [3], the neurodegenerative mechanisms and risk factors in adults are unclear, and little is known regarding the molecular and neurodevelopmental consequences of METH exposure to the fetus. METH abuse during pregnancy has been associated with malformations and other physical defects, including low birth weight, cleft palate, reduced head circumference, and cerebral hemorrhage [4]. Fetal exposure during pregnancy to amphetamines may also predispose these children to altered neurobehavioral function [5]. The increasing abuse of club drugs, including METH, by women of child-bearing age necessitates a fuller understanding of the underlying neurodegenerative mechanisms and risk factors.
Although prenatal exposure to METH leading to postnatal behavioral effects has not been evaluated in mice, one study in rats with twice daily exposure for 6 days found a number of behavioral deficits, including delayed development of locomotion and memory impairment [6]. Multiple daily exposures over 10 days in the postnatal period also resulted in long-term behavioral effects in rats [7].
Cumulative evidence in several adult mammalian models suggests that amphetamine analogs, including METH, are potent and selective neurotoxins that cause neuronal damage through the generation of reactive oxygen species (ROS), either by the redox cycling of catecholamine metabolites ([2] and references therein) or via the direct bioactivation of amphetamines to free radical intermediates catalyzed proximally by brain peroxidases like the prostaglandin H synthases [8]. In the absence of adequate antioxidative cytoprotection or macromolecular repair, ROS can cause irreversible damage to cellular macromolecules such as DNA, RNA, protein, and lipid membranes, or can alter signal transduction pathways, either of which may adversely affect cellular function and contribute to a wide spectrum of pathologies, including altered fetal development [9], [10], [11].
The developing embryo and fetus are substantially deficient in most antioxidative enzymes, and may therefore be at high risk from the embryopathic effects of both endogenous and drug-enhanced oxidative stress and ROS [9], [12]. In the absence of adequate antioxidative enzymes or pathways for the repair of oxidative DNA damage, even endogenous ROS can cause in utero embryonic death and structural birth defects, and such embryopathies are substantially enhanced by ROS-initiating teratogens [9], [13], [14].
Accordingly, we hypothesized that prenatal METH exposure may cause oxidative DNA damage in the embryonic and/or fetal brain, resulting in permanent postnatal neurodevelopmental deficits. This hypothesis was investigated for both the embryonic period of organogenesis (gestational day [GD] 14) and the later fetal period of functional development (GD 17). Unlike the rat studies involving multiple exposures over several days, we determined whether a single low or high dose of METH could enhance ROS formation and oxidative DNA damage, quantified as 8-oxo-2′-deoxyguanosine (8-oxo-dG) formation, in conceptal brain and liver. The potential cellular and functional relevance of this METH-initiated DNA oxidation was evaluated by the consequential effects on postnatal dopaminergic neuronal structure, characterized immunohistochemically by tyrosine hydroxylase staining, and motor coordination, assessed by rotarod performance. These parameters were evaluated from 6 weeks after birth until 3 months of age to distinguish long-term and potentially permanent neurodevelopmental deficits from reversible, receptor-mediated neurotoxicity. Our results provide the first evidence for amphetamine-initiated conceptal oxidative DNA damage leading to long-term postnatal neurodevelopmental deficits, which appear to involve a mechanism different from that for the neurodegenerative effects in amphetamine-exposed adults.
Section snippets
Chemicals
8-Hydroxy-2′-deoxyguanosine was obtained from Cayman Chemical Co. (Ann Arbor, MI), nuclease P1 and Escherichia coli alkaline phosphatase from Sigma-Aldrich (Oakville, ON, Canada), chloroform:isoamyl alcohol:phenol (CIP, 24:1:25) from Life Technologies, Inc. (Burlington, ON, Canada), and proteinase K from Roche Diagnostics (Laval, QC, Canada). All other reagents used were of analytical or HPLC grade.
Drugs
Pure racemic (d/l)-METH was provided by the Healthy Environments and Consumer Safety Branch of
DNA oxidation is increased in METH-exposed GD 14 embryonic brain and liver
Within 1 h in the embryonic brain, compared to saline controls, 20 mg/kg METH caused a 1.6-fold elevation in DNA oxidation (P < 0.003) (Fig. 1, upper panel). Increasing the dose of METH to 40 mg/kg did not further enhance DNA oxidation, although a 1.6-fold increase was still maintained (P < 0.005). By 4 h, oxidative DNA damage in the embryonic brain remained elevated and was unchanged from the 1-h time point for 20 mg/kg (P < 0.02) and 40 mg/kg METH (P < 0.04).
Within 1 h in the embryonic liver,
Discussion
The results from this study provide the first evidence for the enhancement of conceptal DNA oxidation by METH leading to long-term and possibly permanent postnatal neurodevelopmental deficits in motor coordination. Unlike many types of functional deficits, the neurodevelopmental deficits were initiated by a single exposure to METH during either the embryonic period of organogenesis or the later fetal period of functional development, indicating a broad gestational window of risk. The long-term
Acknowledgments
The authors thank Wanda Newerly and Dr. John Roder for their consultations in the behavioral studies, and Kelvin Hui for his technical assistance.
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A preliminary report of this research was presented at the 2003 annual meeting of the Society of Toxicology (USA) [Toxicol. Sci. (Supplement: The Toxicologist), 72 (S-1):342 (No. 1661), 2003]. These studies were supported by a grant from the Canadian Institutes of Health Research (CIHR). W.J. was supported by a doctoral award from the CIHR/Rx&D Health Research Foundation, and the Covance doctoral fellowship from the Society of Toxicology (USA). A.W. was supported by a doctoral award from the Natural Sciences and Engineering Research Council of Canada.
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These authors contributed equally.