Methamphetamine and Lipid Peroxidationin the Rat Retina

Pedro Melo,1,2 Lorena G. Rodrigues,2,3 Maria Dolores Pinazo-Duran,4 and Maria Amelia Tavares1,2*1Institute of Anatomy, Faculty of Medicine, University of Porto, Portugal

2Institute for Molecular and Cell Biology, University of Porto, Portugal3Institute for Biomedical Science Abel Salazar, University of Porto, Portugal

4Ophthalmology Research Unit “Santiago Grisolia,” University Hospital Doctor Peset, Valencia, Spain

Received 11 August 2004; Accepted 27 January 2005

BACKGROUND: The use of psychoactive drugs during adolescence and early adult life has increased in thelast few decades. It is known that developmental exposure to psychostimulants affects the sensory systems, andthe retina has been shown to be a target tissue. This work was conducted to evaluate the pattern of lipidperoxidation in the rat retina following prenatal exposure to methamphetamine (MA). METHODS: Pregnantfemale Wistar rats were given MA (5 mg/kg of body weight/day; SC, in 0.9% saline) from GD 8 to 22.Offspring were sacrificed at postnatal days (PNDs) 7, 14, and 21. The retinas were homogenized, and both thetotal antioxidant and superoxide dismutase (SOD) activities were measured by enzymatic-colorimetric meth-ods. The lipid peroxidation byproducts (malondialdehyde [MDA] and MDA-like metabolites) were measuredby the thiobarbituric acid test. RESULTS: Total antioxidant levels were lower in the MA group at PND 21 inboth males and females. The activity of SOD was higher in PND 7 females from the MA group. MDA levelswere higher in the MA group at PND 21 in both genders. CONCLUSIONS: These findings suggest thatprenatal-induced MA toxicity in the retina may be related to lipid peroxidation processes and oxidative stress.Birth Defects Research (Part A) 73:455–460, 2005. © 2005 Wiley-Liss, Inc.

Key words: rat; retina; lipid peroxidation; oxidative stress; methamphetamine

INTRODUCTION

It is well known that the development of the centralnervous system (CNS) is adversely affected by exposure totoxic agents, including environmental contaminants, in-dustrial toxins, food preservatives, pharmacological sub-stances, drugs of abuse, and alcohol (Aust et al., 1993).Although the mechanisms of action of these diverse agentsat the cellular and molecular levels are poorly understood,there is emerging evidence that the peroxidases and cyto-chrome P450s play an important role in the oxidative bio-transformation of a great variety of exogenous and endog-enous lipophilic toxic compounds (Buhler et al., 1992; Austet al., 1993).

Amphetamines, and in particular MA, are drugs ofabuse that act indirectly in the sympathomimetic system,causing a massive release of dopamine in the brain bysimultaneously preventing the degradation of dopamineby inhibiting both monoamine oxidase activity and do-pamine uptake (Graham, 1978; Giros et al., 1996; Golem-biowska and Zylewska, 1998). However, some of theneurological complications related to exposure to psy-chostimulants have also been attributed to dopamine-

mediated vasoconstrictive effects (Wang et al., 1990;Volkow et al., 1997). Dopamine reacts with molecular ox-ygen to form reactive oxygen species, such as the super-oxide and hydroxyl free radicals, and hydrogen peroxide(Graham, 1978). It is known that chronic administration ofMA in 60-day-old rats causes a long-term deficit in thedopamine system (Pu and Vorhees, 1993) and has a mod-erate persistent effect in 20-day-old rats (Kokoshka et al.,2000). In adults, MA induces an acute massive release ofserotonin, followed by reduction of the synthesis and me-tabolism of serotonin and its transporters (Frost and Cadet,

Grant sponsor: Sponsoring program from Fundacia Ciencia e Tecnologia(PRAXIS); Grant numbers: XXI/BD/3395/2000 (to P.M.), XXI/BD/18508/98 (toL.G.R.); Grant sponsor: Instituto de Salud Carlos III, Programa de Financia-mento Plurianual do Instituto Biologia Molecular e Celular (IBMC) (to M.D.P.-D.); Grant sponsor: Instituto de Salud Carlos III, Fundo Europeu Desenvolvi-mento Regional (FIS-FEDER); Grant number: P10 20191 (to M.D.P.-D.).*Correspondence to: Maria Amelia Tavares, Institute of Anatomy, Faculty ofMedicine of the University of Porto, Alameda Hernani Monteiro, 4200-319Porto, Portugal. E-mail: anatclin@med.up.ptPublished online 6 May 2005 in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/bdra.20138

© 2005 Wiley-Liss, Inc. Birth Defects Research (Part A) 73:455–460 (2005)

Birth Defects Research (Part A): Clinical and Molecular Teratology 73:455–460 (2005)

2000). It is also known that MA can exacerbate the effect ofother toxic substances; for example, it enhances kainicacid–induced toxicity in the adult rat retina (Rodrigues etal., 2004).

MA is increasingly being used as a drug of abuse. Dur-ing the 1990s, it was estimated that approximately one-third of MA users were pregnant women (Vega et al.,1993). This use of MA, and other psychostimulants, bypregnant women has led several research groups to inves-tigate, using a variety of experimental models, the effectsof this psychotropic substance on the perinatal develop-ment of the offspring (Silva-Araujo et al., 1991; Silva-Araujo and Tavares, 1995; Tavares et al., 1996; Gomes-da-Silva et al., 1998; Frost and Cadet, 2000; Summavielle et al.,2000, 2002). The results of the different studies are notalways in agreement and, as a consequence, knowledgeabout the effects of prenatal exposure to MA is still limited.The reasons for the varying results are probably related todifferences in experimental models and animal strainsused, doses of MA administered, and the period of timethe animals were exposed to the drug.

In humans, the use of MA during pregnancy is associ-ated with developmental defects in children, including lowbirth weight, cleft palate, reduced head circumference, andcerebral hemorrhage (Plessinger, 1998; Smith et al., 2001,2003). Children who have been exposed prenatally to am-phetamine may exhibit neurobehavioral alterations such asslowed learning, impaired performance on psychometrictests, aggressive behavior, and poor social adjustment(Cernerud et al., 1996; Eriksson et al., 2000a, 2000b).

It is well known that the developing visual system isextremely vulnerable to the effects of prenatal exposure toneuroactive drugs (Dominguez et al., 1991; Good et al.,1992). Eye defects, including anophthalmia and folded ret-ina, have been reported to occur in rodent offspring afterearly and late prenatal exposure to MA, respectively(Acuff-Smith et al., 1996).

Insight into the extent of MA-induced early gestationallesions can be gained from study of the developing visualsystem during the neonatal period, since, within the neo-natal period, CNS structures appear to be particularly vul-nerable to toxic insults. Taking these facts into account, andconsidering that reactive oxygen species (ROS) are associ-ated with several ocular pathologies (Anderson et al., 1994;Qi et al., 2003) and that drugs such as alcohol can generatefree radicals during their metabolism (Stromland andPinazo-Duran, 2002), we have studied the processes oflipid peroxidation in the retina after prenatal exposure toMA. In order to gain an understanding into whether MAaffects lipid peroxidation in the developing retina, wemade use of a well-controlled experimental model that hasbeen used in a number of previous studies by a variety ofauthors (Silva-Araujo et al., 1995a, 1995b, 1996; Silva-Araujo and Tavares, 1996; Verdejo et al., 1999; Pinazo-Duran et al., 2000).

MATERIALS AND METHODSExperimental Design and Drug AdministrationThe animals used in this study were Wistar rats bred in

the Institute for Molecular and Cell Biology, Porto, Portu-gal, under the institutional guidelines for animal care. Nul-liparous female rats, approximately 2 months old, werehoused under conditions of constant temperature and hu-midity with a 12-hr light/dark cycle. These females were

caged overnight with males and checked for sperm thenext morning. The presence of copulatory plugs or spermin the vaginal cytology was considered as an indication ofpregnancy (GD 1).

The experimental model used in the current study fol-lowed previous reports on the experimental exposure topsychostimulants (Silva-Araujo et al., 1995a, 1995b, 1996;Silva-Araujo and Tavares, 1996; Gomes-da-Silva et al.,2002).

Nine dams were used for each treatment (control andMA) totaling 18 animals for the experiment. In summary,pregnant females were separated and randomly assignedto the different groups at GD 8. Pregnant rats of MAgroups were injected subcutaneously with a total of 5mg/kg of body weight/day of d-N, �-dimethylphenethyl-amine (Sigma-Aldrich, St. Louis, MO), in 0.9% saline, fromGD 8 to 22. Both the schedule and volume for injectingsaline in the pair-fed control group were the same as forthe MA dams. In the pair-fed control group, the amount offood that the MA group consumed in the same gestationalday was similar to that of the MA group (Silva-Araujo etal., 1995a, 1995b, 1996; Silva-Araujo and Tavares, 1996).After delivery (PND 0), each litter was randomly assignedto the 3 different postnatal day ages (PND 7, 14, and 21).

Maternal and Litter VariablesChanges in maternal body weight and food consump-

tion during pregnancy were recorded, as well as theweight of each pup the day after delivery (PND 1). At PND1, the litters were culled to 8 pups: 4 males and 4 females.Animals were identified by painting their paws, head, ortrunk with indelible ink according to a coding system. Thepups were weighed everyday at 9:00 am to avoid variation.

Maternal weight gain was determined by calculating thedifference between body weight each day, from GD 8 to 22(Spear et al., 1989; Silva et al., 1995). The body weight of thepups was recorded from PND 1 to 21.

Assay ProtocolsAt the end of each experimental group (PND 7, 14, and

21), both control and MA-exposed rats were sacrificed. Theeyeballs were enucleated and weighed, and the retinaswere dissected on ice in dim light and stored at �70°C. Theretinas from pups of the same age, gender, and litter werehomogenized together in 20 mM Tris buffer (1/10 pervolume) using an Ultra Turrax (Gravimetria, Porto, Portu-gal).

The protein content of each sample was measured by amodified Lowry kit for protein determination from Sigma-Aldrich (Lowry et al., 1951). Total antioxidant activity andsuperoxide dismutase (SOD) activity were measured byenzymatic-colorimetric commercial kits (Randox Labs, Bar-celona, Spain). The lipid peroxidation activity was mea-sured by the thiobarbituric acid (TBA) test.

Total antioxidant activity. For measurement of total an-tioxidant activity in the retina, 2,2�-azino-di-[3-ethylbenz-thiazoline sulfonate] was incubated with a peroxidase(metmyoglobin) and H2O2 to produce the radical cation2,2�-azino-di-[3-ethylbenzthiazoline sulfonate] that has arelatively stable blue-green color, which can be measuredat 600 nm. The antioxidants present in the sample cause areduction of the color production proportional to theirconcentration (Rice-Evans and Miller, 1994; McLauchlan etal., 1998).

456 MELO ET AL.

Birth Defects Research (Part A) 73:455–460 (2005)

SOD activity. For the measurement of SOD activity inthe retina, xanthine and xanthine oxidase were added inorder to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chlo-ride to form a red formazan dye. The SOD activity wasmeasured by the reduction of this reaction (Verdejo et al.,1999).

TBA test. The lipid peroxidation activity (malondialde-hyde-like metabolites, MDA) in the retina was measuredby the TBA test. In brief, to 125 �l of sample, 125 �l of PBS,1 ml of 0.1 mM HCl, and 100 �l of sodium dodecyl sul-phate (SDS), 150 �l of 1% phosphotungstic acid, and 300 �lof TBA were added. Samples were boiled for 60 min andthe reaction stopped on ice. Subsequently, 1 ml of n-buta-nol was added, and samples were centrifuged at 2800 rpmfor 10 min at 25°C and the fluorescence was read at 544–590 nm (Verdejo et al., 1999).

StatisticsAll comparisons among groups were performed using

2-way ANOVA. When differences were detected, compar-isons were made using the Tukey honest significant differ-ences (HSD) test for unequal n. Confidence intervals weretaken at 95%. Statistics were carried out using the softwarepackage Statistica 5.0 (StatSoft, Tulsa, OK).

RESULTSAnimal Model

Both the maternal body weight increment during preg-nancy and the body weight development of the offspringin the first 21 days of postnatal life did not differ betweencontrol and MA groups.

The mean eyeball weights did not show significant dif-ferences between the control and MA groups at all mea-sured ages (PND 7, 14, 21); however, although there wereno significant differences, in the females of the MA groups,the weight of the eyeballs was lower at all ages comparedwith the respective control groups (Fig. 1).

Lipid PeroxidationThe total level of antioxidants in the retina decreased

with age, for both males and females. The total level of

antioxidants in the MA female group, at PND 7, wassignificantly lower than its control (p � 0.05). At PND 14,the total level of antioxidants in females from the MAgroup was significantly higher than the control group fromthe same age (p � 0.05). In males, the total antioxidant levelwas somewhat higher, although this was not statisticallysignificant, in the MA group both at PND 7 and 14. Thetotal antioxidant level was lower, although again not sig-nificantly so, for both males and females at PND 21 in theMA group, relative to controls (Fig. 2).

SOD activity was significant higher in the MA group atPND 7 for females (p � 0.05). No other differences betweenthe MA and control groups were found. As has beenshown in other experiments (Yamashita et al., 1994; Ogawaet al., 1997), an age-related increase for both males andfemales, was found (Fig. 3).

The levels of malondialdehyde-like metabolites weresignificantly higher in the MA group at PND 21 for females(p � 0.05). No other significant differences were observedfor these parameters (Fig. 4).

Figure 1. Development of eyeball weight of prenatally MA-ex-posed (Male MA and Female MA) rats and respective controls(Male C and Female C). Values are means (SD bars have beenremoved for clarity).

Figure 2. Total antioxidant levels in the rat retina at PND 7, 14,and 21 in MA prenatally-exposed animals (Male MA and FemaleMA) and respective age-matched control groups (Male C andFemale C). Values are means � SD; *p � 0.05.

Figure 3. Superoxide dismutase activity levels in the rat retina atPND 7, 14, and 21 in MA prenatally-exposed animals (Male MAand Female MA) and respective age-matched control groups(Male C and Female C). Values are means � SD; *p � 0.05.

457MA AND LIPID PEROXIDATION IN THE RAT RETINA

Birth Defects Research (Part A) 73:455–460 (2005)

DISCUSSION

This work evaluated oxidative stress in the rat visualsystem as a response to a toxic insult of MA. Previously,others have studied oxidative stress with respect to theeffect of other toxic substances in the visual system (Veriacet al., 1993; Babizhayev and Costa, 1994; Harris et al., 2000;Pinazo-Duran et al., 2000). Many xenobiotics, such as en-vironmental pollutants, pharmacological substances, andalcohol, induce free radical formation in the developingretina (Stromland and Pinazo-Duran, 2002); however, priorto this study, no information was available regardingwhether early exposure to MA also causes lipid peroxida-tion to occur in the developing retina. In the adult mousebrain, it has been shown that MA can cause a severeperturbation in antioxidant enzyme activities and a signif-icant increase in lipid peroxidation processes (Jayanthi etal., 1998).

With regard to the animal model used, it is important torecognize that although the food intake of pregnant fe-males of the control group was controlled (i.e., animalswere pair-fed with the correspondent MA group), asmaller increase of body weight gain was observed in thecontrol groups; however, the difference was not statisti-cally significant and pair-feeding is a valid control used byothers (Tilson, 1992; Spear and Heyser, 1993; Gomes-da-Silva et al., 2002). Moreover, this experimental design hasbeen frequently used for evaluation of the visual system(Silva-Araujo et al., 1995a, 1995b, 1996; Silva-Araujo andTavares, 1996).

In agreement with previous studies, we did not find anydifferences in weight development of offspring betweencontrol and MA groups from birth until PND 21 (Gomes-da-Silva et al., 2002). Other authors have described a retar-dation of eye weight gain when rats are exposed to alcoholduring the early period of development (Pinazo-Duran etal., 1993; Stromland and Pinazo-Duran, 1994). In the cur-rent study, we failed to detect significant differences be-tween control and MA groups; nevertheless, the eyeballsdid consistently weigh less in the MA female groups, sug-gesting a possible delayed eye development induced bythe treatment, which might have been more clear-cut witha greater experimental number.

As a methodology strategy, we had to collect the retinasof the different animals in each group at each age, alltogether, to be able to quantify the different parameters,despite the problem of oversampling that can occur (Hol-son and Pearce, 1992).

We found a gender-related difference concerning theeffect of MA on lipid peroxidation in the rat retina; thus,females appeared more affected than males. A similar phe-nomenon has been observed with other developmentalparameters such as tyrosine hydroxylase mRNA levels infemales prenatally exposed to MA (Gomes-da-Silva et al.,2002). The lipid peroxidation activity (indicating ROS for-mation) was proven by the higher levels of MDA in MA-exposed PND 21 females, which fits with more permanenteffects of MA throughout development and retinal matu-ration. In a previous study of the effects of the pre- andpostnatal exposure to alcohol on free radical formation, asignificant increase in MDA in the rat brain was also reg-istered (Muresan and Eremia, 1997). In the present work,the total antioxidant activity was shown to change signif-icantly between groups, specifically for females at PND 7and 14. At PND 21, levels were lower in the MA group ofboth genders. This result may be explained by a morepermanent effect of MA on retinal development. Theseeffects may underlie the enhanced neurotoxic effect ofexposure to MA in adulthood followed by prenatal expo-sure to MA (Heller et al., 2001a, 2001b).

In this study, the evolution of SOD activity with agefollows the same patterns as in other studies. The age-related increase by the day of eye opening (approximatelyPND 14) is probably induced by light exposure (Yamashitaet al., 1994; Ogawa et al., 1997). A study performed in adultmice reported a decrease of SOD activity in several regionsof the brain (frontal cortex, caudate putamen, and hip-pocampus) after ecstasy exposure (Jayanthi et al, 1999).Our data, however, suggest that MA does not affect theactivity of SOD in the developing rat retina. In other ex-periments, such as when rats were prenatally exposed tothe heavy metal lead, a decrease of SOD activity in hypo-thalamus was observed in 23-day-old Wistar rats (Moreiraet al., 2001), although it should be stated that no differencesbetween controls and animals prenatally treated with leadwere observed when rats were analyzed in adulthood(Moreira et al., 2001). In another study of acute postnatalexposure to alcohol, the levels of SOD in the cerebellumwere enhanced when compared with the control group(Heaton et al., 2002).

Although the cellular and molecular events involved inMA-induced neurotoxicity remain to be elucidated, thedata obtained in this work globally suggest that exposureto MA during pregnancy induces changes in eye and ret-inal development, probably by biochemical mechanismsand oxidative stress. Oxidative stress has been suggestedas one of the prime candidates for these events (Wagner etal., 1980; De Vito and Wagner, 1989; Cadet and Brannock,1998; Yamamoto and Zhu, 1998). It seems that the toxicityof MA, and other substituted amphetamines, involves acascade of events that includes the production of hydrogenperoxide, superoxide, and also hydroxyl radicals (Gio-vanni et al., 1995; Fumagalli et al., 1999; Jayanthi et al.,1999). This pattern of neurodevelopmental toxicity in theretina may be related to lipid peroxidation processes thatrequire further research to be fully elucidated.

Figure 4. Malondialdehyde-like metabolites-MDA levels in therat retina at PND 7, 14, and 21 in MA prenatally-exposed animals(Male MA and Female MA) and respective age-matched controlgroups (Male C and Female C). Values are means � SD; *p � 0.05.

458 MELO ET AL.

Birth Defects Research (Part A) 73:455–460 (2005)

ACKNOWLEDGMENTSWe thank Dr. Glyn Chidlow, from the Nuffield Labora-

tory of Ophthalmology, Oxford (U.K.), for the Englishreview of the manuscript and Dr. Enrique Sevilla-Romero,Valencia (Spain), for technical assistance.

REFERENCESAcuff-Smith KD, Schilling MA, Fisher JE, Vorhees CV. 1996. Stage-specific

effects of prenatal d-methamphetamine exposure on behavioral andeye development in rats. Neurotoxicol Teratol 18:199–215.

Anderson RE, Kretzer FL, Rapp LM. 1994. Free radicals and ocular disease.Adv Exp Med Biol 366:73–86.

Aust SD, Chignell CF, Bray TM, et al. 1993. Free radicals in toxicology.Toxicol Appl Pharmacol 120:168–178.

Babizhayev MA, Costa EB. 1994. Lipid peroxide and reactive oxygen speciesgenerating systems of the crystalline lens. Biochim Biophys Acta 1225:326–337.

Buhler R, Lindros KO, Nordling A, et al. 1992. Zonation of cytochrome P450isozyme expression and induction in rat liver. Eur J Biochem 204:407–412.

Cadet JL, Brannock C. 1998. Free radicals and the pathobiology of braindopamine systems. Neurochem Int 32:117–131.

Cernerud L, Eriksson M, Jonsson B, et al. 1996. Amphetamine addictionduring pregnancy: 14-year follow-up of growth and school perfor-mance. Acta Paediatr 85:204–208.

De Vito MJ, Wagner GC. 1989. Methamphetamine-induced neuronal dam-age: a possible role for free radicals. Neuropharmacology 28:1145–1150.

Dominguez R, Aguirre Vila-Coro A, Slopis JM, Bohan TP. 1991. Brain andocular abnormalities in infants with in utero exposure to cocaine andother street drugs. Am J Dis Child 145:688–695.

Eriksson M, Jonsson B, Steneroth G, Zetterstrom R. 2000a. Amphetamineabuse during pregnancy: environmental factors and outcome after14-15 years. Scand J Public Health 28:154–157.

Eriksson M, Jonsson B, Zetterstrom R. 2000b. Children of mothers abusingamphetamine: head circumference during infancy and psychosocialdevelopment until 14 years of age. Acta Paediatr 89:1474–1478.

Frost DO, Cadet JL. 2000. Effects of methamphetamine-induced neurotox-icity on the development of neural circuitry: a hypothesis. Brain ResBrain Res Rev 34:103–118.

Fumagalli F, Gainetdinov RR, Wang YM, et al. 1999. Increased metham-phetamine neurotoxicity in heterozygous vesicular monoamine trans-porter 2 knock-out mice. J Neurosci 19:2424–2431.

Giovanni A, Liang LP, Hastings TG, Zigmond MJ. 1995. Estimating hy-droxyl radical content in rat brain using systemic and intraventricularsalicylate: impact of methamphetamine. J Neurochem 64:1819–1825.

Giros B, Jaber M, Jones SR, et al. 1996. Hyperlocomotion and indifference tococaine and amphetamine in mice lacking the dopamine transporter.Nature 379:606–612.

Golembiowska K, Zylewska A. 1998. N6-2-(4-aminophenyl)ethyladenosine(APNEA), a putative adenosine A3 receptor agonist, enhances meth-amphetamine-induced dopamine outflow in rat striatum. Pol J Phar-macol 50:299–305.

Gomes-da-Silva J, Silva MC, Tavares MA. 1998. Developmental exposure tomethamphetamine: a neonatal model in the rat. Ann NY Acad Sci844:310–313.

Gomes-da-Silva J, Perez-Rosado A, de Miguel R, et al. 2002. Prenatal expo-sure to methamphetamine in the rat: ontogeny of tyrosine hydroxylasemRNA expression in mesencephalic dopaminergic neurons. Ann NYAcad Sci 965:68–77.

Good WV, Ferriero DM, Golabi M, Kobori JA. 1992. Abnormalities of thevisual system in infants exposed to cocaine. Ophthalmology 9:341–346.

Graham DG. 1978. Oxidative pathways for catecholamines in the genesis ofneuromelanin and cytotoxic quinones. Mol Pharmacol 14:633–643.

Harris SJ, Wilce P, Bedi KS. 2000. Exposure of rats to a high but not low doseof ethanol during early postnatal life increases the rate of loss of opticnerve axons and decreases the rate of myelination. J Anat 197(Pt3):477–485.

Heaton MB, Madorsky I, Paiva M, Mayer J. 2002. Influence of ethanol onneonatal cerebellum of BDNF gene-deleted animals: analyses of effectson Purkinje cells, apoptosis-related proteins, and endogenous antioxi-dants. J Neurobiol 5:160–176.

Heller A, Bubula N, Freeney A, Won L. 2001a. Elevation of fetal dopaminefollowing exposure to methamphetamine in utero. Brain Res Dev BrainRes 130:139–142.

Heller A, Bubula N, Lew R, Heller B, Won L. 2001b. Gender-dependentenhanced adult neurotoxic response to methamphetamine followingfetal exposure to the drug. J Pharmacol Exp Ther 298:769–779.

Holson RR, Pearce B. 1992. Principles and pitfalls in the analysis of prenatal

treatment effects in multiparous species. Neurotoxicol Teratol 14:221–228.

Jayanthi S, Ladenheim B, Cadet JL. 1998. Methamphetamine-inducedchanges in antioxidant enzymes and lipid peroxidation in copper/zinc-superoxide dismutase transgenic mice. Ann NY Acad Sci 844:92–102.

Jayanthi S, Ladenheim B, Andrews AM, Cadet JL. 1999. Overexpression ofhuman copper/zinc superoxide dismutase in transgenic mice attenu-ates oxidative stress caused by methylenedioxymethamphetamine (Ec-stasy). Neuroscience 91:1379–1387.

Kokoshka JM, Fleckenstein AE, Wilkins DG, Hanson GR. 2000. Age-depen-dent differential responses of monoaminergic systems to high doses ofmethamphetamine. J Neurochem 75:2095–2102.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measure-ment with the Folin phenol reagent. J Biol Chem 193:265–275.

McLauchlan WR, Sanderson J, Quinlan M, Williamson G. 1998. Measure-ment of the total antioxidant activity of human aqueous humor. ClinChem 44:888–889.

Moreira EG, Rosa GJ, Barros SB, et al. 2001. Antioxidant defense in rat brainregions after developmental lead exposure. Toxicology 169:145–151.

Muresan C, Eremia I. 1997. Ethanol stimulates the formation of free oxygenradicals in the brain of newborn rats. Rom J Morphol Embryol 43:113–117.

Ogawa T, Ohira A, Amemiya T. 1997. Manganese and copper-zinc super-oxide dismutases in the developing rat retina. Acta Histochem 99:1–12.

Pinazo-Duran MD, Renau-Piqueras J, Guerri C. 1993. Developmentalchanges in the optic nerve related to ethanol consumption in pregnantrats: analysis of the ethanol-exposed optic nerve. Teratology 48:305–322.

Pinazo-Duran MD, Verdejo C, Azorin I, et al. 2000. Colocalization of alde-hyde dehydrogenases and Fe/NADPH-induced lipid peroxidation intissue sections of rat retina. Ophthalmic Res 32:61–68.

Plessinger MA. 1998. Prenatal exposure to amphetamines. Risks and ad-verse outcomes in pregnancy. Obstet Gynecol Clin North Am 25:119–138.

Pu C, Vorhees CV. 1993. Developmental dissociation of methamphetamine-induced depletion of dopaminergic terminals and astrocyte reaction inrat striatum. Brain Res Dev Brain Res 72:325–328.

Qi X, Lewin AS, Hauswirth WW, Guy J. 2003. Optic neuropathy induced byreductions in mitochondrial superoxide dismutase. Invest OphthalmolVis Sci 44:1088–1096.

Rice-Evans C, Miller NJ. 1994. Total antioxidant status in plasma and bodyfluids. Methods Enzymol 234:279–293.

Rodrigues LR, Tavares MA, Wood JPM, et al. 2004. Methamphetamineexacerbates the toxic effect of kainic acid in the adult rat retina. Neu-rochem Int 45:1133–1141.

Silva MC, Silva-Araujo A, Abreu S, et al. 1995. Effects of prenatal cocaineexposure on postnatal growth patterns of male Wistar rats. Neurotoxi-col Teratol 17:471–477.

Silva-Araujo A, Salgado-Borges J, Tavares MA. 1991. Morphologicalchanges in the optic nerve after chronic exposure of neonatal rats tococaine and amphetamine. Ophthalmic Res 23:295–302.

Silva-Araujo AL, Tavares MA. 1995. Expression of glial fibrillary acidicprotein in the rat retina after exposure to psychostimulants. Retina15:241–247.

Silva-Araujo A, Abreu-Dias P, Silva MC, Tavares MA. 1995a. Effects ofprenatal cocaine exposure in the photoreceptor cells of the rat retina.Mol Neurobiol 11:77–86.

Silva-Araujo A, Silva MC, Abreu-Dias P, Tavares MA. 1995b. Effects ofprenatal cocaine exposure in the retinal ganglion cell layer of the rat. Amorphometric analysis. Mol Neurobiol 11:87–97.

Silva-Araujo A, Tavares MA. 1996. Development of the eye after gestationalexposure to cocaine. Vascular disruption in the retina of rats andhumans. Ann NY Acad Sci 801:274–288.

Silva-Araujo A, Silva MC, Simon A, et al. 1996. The effects of prenatalexposure to cocaine on the dopaminergic cells in the rat retina. Animmunocytochemical and neurochemical study. Exp Eye Res 62:697–708.

Smith LM, Chang L, Yonekura ML, et al. 2001. Brain proton magneticresonance spectroscopy in children exposed to methamphetamine inutero. Neurology 57:255–260.

Smith L, Yonekura ML, Wallace T, et al. 2003. Effects of prenatal metham-phetamine exposure on fetal growth and drug withdrawal symptomsin infants born at term. J Dev Behav Pediatr 24:17–23.

Spear LP, Kirstein CL, Frambes NA. 1989. Cocaine effects on the developingcentral nervous system: behavioral, psychopharmacological, and neu-rochemical studies. Ann NY Acad Sci 562:290–307.

Spear LP, Heyser CJ. 1993. Is use of a cellulose-diluted diet a viable alter-native to pair-feeding? Neurotoxicol Teratol 15:85–89.

Stromland K, Pinazo-Duran MD. 1994. Optic nerve hypoplasia: compara-

459MA AND LIPID PEROXIDATION IN THE RAT RETINA

Birth Defects Research (Part A) 73:455–460 (2005)

tive effects in children and rats exposed to alcohol during pregnancy.Teratology 50:100–111.

Stromland K, Pinazo-Duran MD. 2002. Ophthalmic involvement in the fetalalcohol syndrome: clinical and animal model studies. Alcohol Alcohol37:2–8.

Summavielle T, Silva-Araujo A, Silva MC, Tavares MA. 2000. Effects ofneonatal exposure to cocaine in the development of the neurotransmit-ters retinal systems: an immunocytochemical and neurochemical study.Ann NY Acad Sci 914:418–430.

Summavielle T, Magalhaes A, Castro-Vale I, et al. 2002. Neonatal exposureto cocaine: altered dopamine levels in the amygdala and behavioraloutcomes in the developing rat. Ann NY Acad Sci 965:515–521.

Tavares MA, Silva MC, Silva-Araujo A, et al. 1996. Effects of prenatalexposure to amphetamine in the medial prefrontal cortex of the rat. IntJ Dev Neurosci 14:585–596.

Tilson HA. 1992. Study design considerations in developmental neurotoxi-cology. Neurotoxicol Teratol 14:199–203.

Vega WA, Kolody B, Hwang J, Noble A. 1993. Prevalence and magnitude ofperinatal substance exposures in California. N Engl J Med 329:850–854.

Verdejo C, Marco P, Renau-Piqueras J, Pinazo-Duran MD. 1999. Lipidperoxidation in proliferative vitreoretinopathies. Eye 13:183–188.

Veriac S, Tissie G, Bonne C. 1993. Oxygen free radicals adversely affect theregulation of vascular tone by nitric oxide in the rabbit retina underhigh intraocular pressure. Exp Eye Res 56:85–88.

Volkow ND, Wang GJ, Fowler JS, et al. 1997. Decreased striatal dopaminer-gic responsiveness in detoxified cocaine-dependent subjects. Nature38:830–833.

Wagner GC, Ricaurte GA, Johanson CE, et al. 1980. Amphetamine inducesdepletion of dopamine and loss of dopamine uptake sites in caudate.Neurology 30:547–550.

Wang AM, Suojanen JN, Colucci VM, et al. 1990. Cocaine- and metham-phetamine-induced acute cerebral vasospasm: an angiographic studyin rabbits. AJNR Am J Neuroradiol 11:1141–1146.

Yamamoto BK, Zhu W. 1998. The effects of methamphetamine on theproduction of free radicals and oxidative stress. J Pharmacol Exp Ther287:107–114.

Yamashita H, Horie K, Yamamoto T, et al. 1994. Superoxide dismutase indeveloping mouse retina. Jpn J Ophthalmol 38:148–161.

460 MELO ET AL.

Birth Defects Research (Part A) 73:455–460 (2005)