Metallothionein Inducers Protect Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Neurotoxicity in Mice

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a drug that induces parkinsonism in human and non-human primates. Free radicals are thought to be involved in its mechanism of action. Recently, the participation of metallothionein as scavenger of free radicals has been proposed. In this work, we studied the effect of metallothionein inducers in MPTP neurotoxic action. Male swiss albino mice were pretreated either with cadmium (1 mg/kg) or dexamethasone (5 mg/kg), two well-known inducers of metallothionein synthesis, and 5 hours later with an MPTP administration (30 mg/kg). Treatment schedule was repeated daily for either 3 or 5 consecutive days. All animals were killed 7 days after the last administration, and striatal dopamine and homovanillic acid contents were analyzed as an end-point of MPTP neurotoxicity. Striatal dopamine content of cadmium plus MPTP-treated animals (3-days) increased by 32%, and 48% (5-days) vs MPTP-alone animals. Dexamethasone plus MPTP-treated group also showed increased dopamine levels 28% (3-days) and 43% (5-days). MPTP treatment reduced striatal metallothionein concentration (49% vs control animals). Dexamethasone and cadmium increased metallothionein concentrations in MPTP-treated groups, by 77% and 82% respectively. Results suggest that metallothionein induction provide a significant resistance factor against the deleterious effect of MPTP.     

S-Allylcysteine, a garlic-derived antioxidant, ameliorates quinolinic acid-induced neurotoxicity and oxidative damage in rats

Excitotoxicity elicited by overactivation of N-methyl-D-aspartate receptors is a well-known characteristic of quinolinic acid-induced neurotoxicity. However, since many experimental evidences suggest that the actions of quinolinic acid also involve reactive oxygen species formation and oxidative stress as major features of its pattern of toxicity, the use of antioxidants as experimental tools against the deleterious effects evoked by this neurotoxin becomes more relevant. In this work, we investigated the effect of a garlic-derived compound and well-characterized free radical scavenger, S-allylcysteine, on quinolinic acid-induced striatal neurotoxicity and oxidative damage. For this purpose, rats were administered S-allylcysteine (150, 300 or 450 mg/kg, i.p.) 30 min before a single striatal infusion of 1 microl of quinolinic acid (240 nmol). The lower dose (150 mg/kg) of S-allylcysteine resulted effective to prevent only the quinolinate-induced lipid peroxidation (P < 0.05), whereas the systemic administration of 300 mg/kg of this compound to rats decreased effectively the quinolinic acid-induced oxidative injury measured as striatal reactive oxygen species formation (P < 0.01) and lipid peroxidation (P < 0.05). S-Allylcysteine (300 mg/kg) also prevented the striatal decrease of copper/zinc-superoxide dismutase activity (P < 0.05) produced by quinolinate. In addition, S-allylcysteine, at the same dose tested, was able to reduce the quinolinic acid-induced neurotoxicity evaluated as circling behavior (P < 0.01) and striatal morphologic alterations. In summary, S-allylcysteine ameliorates the in vivo quinolinate striatal toxicity by a mechanism related to its ability to: (a) scavenge free radicals; (b) decrease oxidative stress; and (c) preserve the striatal activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). This antioxidant effect seems to be responsible for the preservation of the morphological and functional integrity of the striatum.     

Dopamine Transporter Is Required for In Vivo MPTP Neurotoxicity: Evidence from Mice Lacking the Transporter

Abstract: The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29-fold increase in the level of GFAP mRNA in the striatum of wild-type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP⁺; 10 m M ), via a microdialysis probe produced a massive efflux of DA in wild-type mice (>320-fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.     

The effect of subchronic, intermittent L-DOPA treatment on neuronal nitric oxide synthase and soluble guanylyl cyclase expression and activity in the striatum and midbrain of normal and MPTP-treated mice

We have investigated the effects of low (10 mg/kg) and high (100 mg/kg) doses of L-DOPA on the expression and activity of neuronal nitric oxide synthase (nNOS) and guanylyl cyclase (GC) in the striatum and midbrain of mice. L-DOPA was administered subchronically for 11 days (beginning 3 days after last MPTP/NaCl injection) or for 14 days (with dosing started immediately following the last MPTP/NaCl injection). Adult mice received three intraperitoneal (i.p.) injections of physiological saline or MPTP at 2h intervals (total dose of 40 mg/kg). Normal and MPTP-injected mice were treated twice a day for 11 or 14 days with low (10/2.5 mg/kg bw) or high (100/25mg/kg bw) doses of L-DOPA/benserazide. The present study indicates that several days of treatment with L-DOPA does not affect MPTP-activation of the nNOS/sGC/cGMP pathway or the neurodegenerative processes that occur in the striatum and midbrain of mice. In normal mice, L-DOPA upregulates the expression and activity of nNOS and GC to levels found in MPTP-injected mice. Due to upregulation of nNOS and GC, cGMP levels in the mouse striatum and midbrain are also elevated, however, significantly lower in mice administrated with low dose of L-DOPA. In both investigated brain regions of normal mice cGMP-dependent PDEs activities were elevated after low dose administration of L-DOPA, but no change in PDEs activities has been detected in MPTP and high L-DOPA-injected mice as compared to control values. The enhancement of nNOS mRNA and GCbeta1 mRNA levels were generated by both doses of L-DOPA, given in a time-dependent fashion. L-DOPA-injected for 11 or 14 days caused a decrease in TH protein levels in the striatum and midbrain, respectively; this result was noted irrespective of dose. L-DOPA therapy did not prevent the MPTP-induced decrease in TH protein levels in either investigated brain region.     

Bak Foong protects dopaminergic neurons against MPTP-induced neurotoxicity by its anti-apoptotic activity

Bak Foong pill (BFP) is a well-known traditional Chinese medicine used for treatment of various gynaecological disorders. In addition, it exerts beneficial effects on other functional systems including the central nervous system. In the present study, we have investigated the possible neuroprotective action of BFP upon the nigrostriatal dopaminergic system by examining its effect on the expression patterns of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the 1-methyl-4-phenyl-1,2,3,6-tetrahyrdropyridine (MPTP)-induced Parkinson's disease (PD) mouse model. MPTP significantly decreased TH and DAT mRNA levels in the striatum and midbrain of both female and male C57BL/6 mice. However, with BFP pre-treatment mice showed a reduced neurotoxicity, with TH and DAT mRNA levels either not affected by MPTP or affected to a lesser extent in the midbrain and striatum when compared to vehicle treated animals. Possible anti-apoptotic activity of BFP was further studied in a dopamine-secreting neuroendocrine cell line, PC12. In this assay, MPTP elevated the expression of a pro-apoptotic gene, Bax, while this expression was reduced by BFP pre-treatment. Flow cytometry results also revealed that the effect of MPTP-induced apoptosis in PC12 cell lines was significantly reduced by BFP. The present results suggest that BFP is able to protect dopaminergic neurons from neurotoxin-induced neuronal injury with anti-apoptotic activity being one of the possible mechanisms.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by ethanol or acetaldehyde

Persistent neurochemical changes consistent with parkinsonism have been reported in brains of mice treated with repeated high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We now report that ethanol or acetaldehyde potentiate MPTP-induced damage to mouse striatum. One hour after the combined treatments (ethanol and MPTP or acetaldehyde and MPTP), the animals exhibited a marked and long-lasting catatonic posture and then returned gradually to apparently normal locomotion. Seven days after MPTP administration, depletion of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mouse striatum were further potentiated in the group of animals treated with ethanol. This effect was more evident when the treatment was repeated twice and was dose-dependent. Acetaldehyde was more potent than ethanol in enhancing MPTP neurotoxicity. A single exposure to acetaldehyde before and during MPTP treatment produced a very consistent fall of DA, DOPAC and HVA but not serotonin (5HT) or 5-hydroxyindoleacetic acid (5HIAA) in the striatum. This suggests that ethanol effects on MPTP neurotoxicity might be related to acetaldehyde formation.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

Protective Role of Quercetin on PCBs-Induced Oxidative Stress and Apoptosis in Hippocampus of Adult Rats

Polychlorinated biphenyls (PCBs) exposure produces neurodegeneration and induces oxidative stress. Neuroprotective role of quercetin, on PCBs induced apoptosis in hippocampus has not yet been studied. The present study is focused to see whether quercetin supplementation precludes against PCBs induced oxidative stress and hippocampal apoptosis. The results have shown that quercetin at 50 mg/kg bwt/30 days has protected oxidative stress in hippocampus of adult male rats. Quercetin, a free radical scavenger decreased the levels of oxidative stress markers in the hippocampus of simultaneous PCB+quercetin treated rats. The pro-apoptotic and anti-apoptotic molecules such as Bad, Bid, Bax and Bcl2 were altered in the hippocampus of experimental animals. PCBs increased the DNA damage and induced neurodegeneration were assessed by histological studies. PCB induced ROS may be linked to increased hippocampal neuronal apoptosis. Quercetin supplementation decreased the neuronal damage and scavenged the free radicals induced by PCBs and protects PCBs induced apoptosis and oxidative stress.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice

S-Allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. 1-Methyl-4-phenylpyridinium (MPP⁺) is the stable metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and it causes nigrostriatal dopaminergic neurotoxicity. Previous studies suggest that oxidative stress, via free radical production, is involved in MPP⁺-induced neurotoxicity. Here, we report on the neuroprotective effect of SAC against oxidative stress induced by MPP⁺ in the striatum of C57BL/6J mice. Mice were pretreated with SAC (125 mg/kg ip) daily for 17 days, followed by administration of MPP⁺ (0.72 mg/kg icv), and were sacrificed 24 h later to evaluate lipid peroxidation, different antioxidant enzyme activities, spontaneous locomotor activity and dopamine (DA) content. MPP⁺ administration resulted in a significant decrease in DA levels in the striatum. Mice receiving SAC (125 mg/kg ip) had significantly attenuated MPP⁺-induced loss of striatal DA levels (32%). The neuroprotective effect of SAC against MPP⁺ neurotoxicity was associated with blocked (100% of protection) of lipid peroxidation and reduction of superoxide radical production — indicated by an up-regulation of Cu-Zn-superoxide dismutase activity — both of which are indices of oxidative stress. Behavioral analyses showed that SAC improved MPP⁺-induced impairment of locomotion (35%). These findings suggest that in mice, SAC attenuates MPP⁺-induced neurotoxicity in the striatum and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

Upregulation of guanylyl cyclase expression and activity in striatum of MPTP-induced parkinsonism in mice

The aim of our study was to investigate the expression and the activity of soluble guanylyl cyclase (GC) and phosphodiesterase (PDE) activities that regulate cGMP level in the striatum, hippocampus, and brain cortex in an animal model of PD, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We observed the increase of total activity and protein level of GC in striatum after MPTP injection. It was accompanied by an enhancement of both mRNA expression and protein level of GCbeta1 subunit. MPTP induces mRNA expression and elevates protein concentration of GCbeta1 in striatum up to 14 days after its injection, which in turn causes a marked enhancement of cGMP formation. Furthermore, the activation of GC occurs through change of maximal enzyme activity (V(max)). Simultaneously, no change in PDE activity has been detected in all investigated regions of the brain after MPTP. MPTP injection caused the elevation of GCbeta1 protein level in both the membrane and cytosol fractions being significantly higher in cytosol. Western blot analysis demonstrated about 45-67% decrease of tyrosine hydroxylase protein content in striatum. These data suggest that NO/cGMP signaling pathway may at least partially contribute to dopaminergic fiber degeneration in the striatum, the damage attributed to PD.     

In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole

The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.     

Systemic Administration of Proteasome Inhibitor Protects Against MPTP Neurotoxicity in Mice

Dysfunction of the proteasome has been suggested to contribute in the degeneration of nigrostriatal dopaminergic neurons. Here, we investigated to determine whether systematic administration of proteasome inhibitor, carbobenzoxy-l-γ-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI) protects against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice. Three administrations of MPTP at 1-h intervals to mice reduced significantly the concentration of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) in the striatum after 5 days. In contrast, PSI (0.3 and 1.0 mg/kg) prevented a significant decrease in dopamine, DOPAC and HVA contents of the striatum 5 days after MPTP treatment. In our Western blot analysis study, PSI at a dose of 1.0 mg/kg prevented a significant decrease in TH (tyrosine hydroxylase) protein and a significant increase in glial fibrillary acidic protein 5 days after MPTP treatment. Furthermore, our immunohistochemical study showed that PSI at a dose of 1.0 mg/kg prevented a significant loss in TH immunopositive neurons in the striatum and substantia nigra 5 days after MPTP treatment. In contrast, PSI caused a significant increase in the number of intense ubiquitin immunopositive cells in the striatum and substantia nigra 5 days after MPTP treatment. These results indicate that proteasome inhibitors can protect against MPTP neurotoxicity in mice. The neuroprotective effect of PSI against dopaminergic cell damage may be mediated by the elevation of ubiquitination. Thus, our findings provide further valuable information for the pathogenesis of Parkinson’s disease. Takuya Oshikawa and Hayato Kuroiwa contributed equally to this work.     

Somatostatin Content Increases Following Norepinephrine Depletion in Frontal Cortex of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mice

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on somatostatin (SS)-containing neurons were examined by measuring dopamine, norepinephrine (NE), SS, and SS mRNA in striatum and frontal cortex of C57/B16 mice at various times following treatment with MPTP-HCl (96 mg/kg i.p.). MPTP caused a 70% depletion of dopamine in striatum by 1 day and a 40% depletion of NE in frontal cortex within 3 days. SS content was increased in frontal cortex 4 days later, but not in striatum; there were no changes in SS mRNA. Maprotiline, a specific NE-uptake blocker, prevented both the depletion of NE and the increase of SS in frontal cortex due to MPTP administration. These results support the possibility that NE can regulate SS in frontal cortex and are discussed in terms of the decrease of SS seen in parkinsonian patients with dementia.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.