Arun L. Jadhav

Lead exposure activates nuclear factor kappa b, activator protein-1, c-Jun N-terminal kinase and caspases in the rat brain

How lead manifests its neurotoxicity is not well understood. The hypothesis that lead may activate nuclear transcription factors NF-kappaB, activator protein-1 (AP-1), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase (MAPKK) and caspases in the rat brain leading to the manifestation of its neurotoxic effects, was tested in 21-day-old male Long-Evans rats exposed to 50 ppm Pb in drinking water for 90 days. After the 90-day exposure, blood lead levels of the rats in control group were 4+/-0.2 microg/dl, while those of the Pb-exposed group were 18+/-0.3 microg/dl (n=50). Similarly, at the end of the exposure period, the Pb-exposed group showed significantly higher accumulation of Pb in brain regions such as, frontal cortex (FC), brain stem (BS), striatum (ST), and hippocampus (HIP) (338.6+/-7.7, 391.6+/-3.8, 288.3+/-6.7, and 382.3+/-3.3 ng/g wet tissue, respectively, in FC, BS, ST, and HIP) than the control group (126.6+/-2.7, 127.6+/-1.8, 201.3+/-9.4, and 180.3+/-4.4 ng/g wet tissue, respectively, in FC, BS, ST, and HIP). There was a 3-4-fold increase in NF-kappaB and AP-1 level in all the four regions of the brain of lead-treated animals. All four regions showed 4-10-fold activation of JNK and a 5-6-fold activation of MAPKK. As indicated by poly(ADP ribose) polymerase cleavage, lead exposure induced the activation of caspases in all four regions. Overall our results indicate that lead exposure induces the activation of NF-kappaB, AP-1, JNK, MAPKK, and caspases in the brain, which may contribute to its neurotoxic effects.

Low level lead exposure decreases in vivo release of dopamine in the rat nucleus accumbens : a microdialysis study

Abstract: The basal and K+‐induced release of dopamine and its metabolites, 3,4‐dihydroxyphenylacetic acid and homovanillic acid, were measured in microdialysate samples obtained in vivo from the nucleus accumbens region of rats subchronically exposed to 50 ppm lead for 90 days. The basal and stimulus‐induced release of dopamine and the metabolites were significantly reduced in the lead‐exposed rats as compared with the controls. These reductions in dopamine and its metabolites are consistent with the reports of decreased dopamine availability associated with lead‐induced changes in certain behavioral indices (fixed‐interval performance) in rats. Furthermore, these changes were observed at blood lead levels similar to those considered to cause impairment in cognitive functions in children.

Contribution of protein kinase C and glutamate in Pb2+-induced cytotoxicity

Activation of protein kinase C (PKC) plays an important role in lead (Pb(2+))-induced cytotoxicity. The effects of low dose exposure to Pb(2+) on cytosolic free calcium (Ca(2+)), PKC activity and mechanisms involved in cell death were studied in PC12 cells. Exposure of PC12 cells to low dose Pb(2+) (0.01 microM) increased PKC activity, while exposure to a higher dose (10 microM) led to decreased PKC activity. Additionally, in normal extracellular medium, low concentration of Pb(2+) (0.01 microM) stimulated increase in cytosolic free calcium while the higher concentrations of Pb(2+) (10 microM) did not. However, the effect of low dose Pb(2+) (0.01 microM) was blocked by removing Ca(2+) from external medium. The role of Pb(2+)-induced changes in PKC activity and its relationship to oxidative stress and related cytotoxicity was also studied. Pb(2+) alone (0.01-10 microM) produced reactive oxygen species (ROS) dose dependently over the period of 24 h. Pb(2+)-induced ROS were potentiated in the presence of 500 microM glutamate. Furthermore, a correlation was observed between ROS generation and the levels of cytotoxicity, which was observed after 24 h exposures to Pb(2+) by trypan blue method, and the cytotoxicity was enhanced by glutamate co-treatment. Pb(2+)-induced cell death was blocked partially by staurosporine (PKC inhibitor, 100 nM) and NMDA antagonist, MK-801 (1 microM). It is concluded that, in Pb-induced cytotoxicity, modulation of PKC and intracellular calcium play significant roles in augmenting glutamate receptor mediated oxidative species formation and subsequent cell death.

PB-INDUCED ALTERATIONS IN TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN

Our previous studies have shown that exposure to low levels of Pb results in significant reductions in dopamine (DA) and its metabolites (3,4-dihyroxyphenylacetic acid, DOPAC and homovanillic acid, HVA) in nucleus acumbens (NA). This area of brain receives dopaminergic projections from the ventral tegmentum and is considered vital in manifestation of many behavioral responses. Similarly, basal and K+-induced release of DA was found significantly reduced in the Pb-exposed rats as compared to the controls in this brain region. Additional studies indicated that acute infusion of Pb in nucleus acumbens caused significant release of DA. Based on these observations it was postulated that the reductions in DA contents and in the basal and stimulus-induced release of DA in NA were manifestations of attenuated dopaminergic activity in this brain region. However, the mechanism of this attenuation is not yet clear. Studies reported here were designed to evaluate the role of a key regulatory enzy me in biosynthesis of DA, i.e. tyrosine hydroxylase (TH) in Pb-induced reductions in dopaminergic activity. The results of these studies indicated that 50 and 500 ppm Pb produced 22.8 and 56% inhibition of TH activity in vitro respectively, and that the enzyme activity was reduced to 43% in rats exposed to 50 ppm lead for 30 days as compared to the controls. The alterations in TH activity in Pb-exposed animals were further confirmed by Western blot analysis. Collectively, these results suggest that Pb-induced inhibition of TH activity in rat brain may contribute to the reductions in dopaminergic activity observed in Pb-exposed animals. (Mol Cell Biochem 175: 137–141, 1997)

Levels of protein kinase C and nitric oxide synthase activity in rats exposed to sub chronic low level lead

The intracellular regulation of protein kinase C (PKC) and nitric oxide synthase (NOS) in relation to the accumulation of lead (Pb2+) in various brain regions following low-level lead exposure (50 ppm) for 90 days in rats was investigated. PKC and NOS are important enzymes in mediating cellular transduction mechanisms and in the regulation of neuronal plasticity. Rats exposed to Pb2+ resulted in bood Pb2+ levels similar to those observed in children affected due to Pb2+ exposure. Further, we examined whether Pb2+ accumulation changed the intracellular signaling mechanisms in different brain regions. Results of these experiments indicate that significant region specific Pb2+ accumulation is associated with down regulation of PKC. The down regulation of PKC increased the activity of NOS following Pb2+ exposure. Thus, the change in PKC activity in respect to Pb2+ accumulation increased NOS activity. These results suggest that neuronal toxicity during Pb2+ exposure is linked to the modulation of PKC followed by NOS activation.

Dietary Sodium Intake and Urinary Dopamine and Sodium Excretion During the Course of Blood Pressure Development in DAHL Salt-Sensitive and Salt-Resistant Rats

Recent studies have suggested that dopamine (DA) formed within the kidney may play an important role in promoting sodium excretion, and that renal production and excretion of DA is determined by dietary sodium intake. Inasmuch as increased sodium consumption produces hypertension in Dahl salt-sensitive (DS) rats but not in Dahl salt-resistant (DR) rats, the present study was designed to examine the relationship between sodium consumption and urinary excretion of DA in these rats. DS and DR rats were placed on either high sodium chloride (8%) or low sodium chloride (0.4%) diets at 4 weeks of age and their systolic blood pressure (SBP), urine volume, urinary sodium and catecholamine excretion were measured once every week for the next 4 weeks. High sodium chloride diet increased SBP in DS rats at 6 weeks of age and SBP continued to rise until they were 8 weeks old. The SBP of DR rats did not reach hypertensive levels when they were given high sodium chloride diet. The SBP of DS rats on low sodium chloride diet was significantly higher than DR rats on the same diet. The urinary DA excretion increased with age in all four groups of rats and was similar when they were 8 weeks old. However, both DS and DR rats on high sodium chloride diet excreted greater amounts of sodium and had increased urine volume compared to the DS and DR rats on low sodium chloride diet. There were no significant differences in urinary NE or E excretion in these four groups of rats. Kidney levels of DA and NE were significantly lower in DS compared to DR rats on high sodium chloride diet. These results show that although there are no differences in urinary DA excretion between rats on low and high sodium intake, both DS and DR rats on high sodium chloride diet are able to exhibit a natriuretic response. The DS rats eliminate sodium at the expense of an elevated SBP whereas DR rats stay normotensive. Therefore, it appears that alterations in mechanisms controlling renal vascular resistance rather than sodium excretion are responsible for the development of hypertension in DS rats.

Changes in mesocorticolimbic dopamine and D1/D2 receptor levels after low level lead exposure: a time course study

Chronic post weaning low-level lead exposure produces cognitive deficits associated with Pb-induced alterations of mesocorticolimbic dopamine (DA) function. This study examined Pb-induced changes in the temporal profile of D1/D2 receptor protein and DA levels in the nucleus accumbens (NAC), hippocampus (HIP), and the frontal cortex (FC). Male Long-Evans rats were exposed to 0 (n=16-20) and 50 ppm Pb (n=16-20) for 180 days. Blood Pb analysis by atomic absorption spectroscopy showed BPb<2 microg/dl in the control group and BPb>9 microg/dl in the Pb-exposed group. Brain DA levels were evaluated by high performance liquid chromatography; D1/D2 receptor expressions, by autoradiographic analysis. Pb exposure produced a transient hyperdopaminergic state, followed by a sustained decline in dopaminergic function within the NAC and a longer-lasting hyperdopaminergic condition within the HIP, whereas it decreased FC D1/D2 without significantly affecting FC DA levels. These findings indicate that time plays a critical, region-specific role in Pbs effects on the normal synaptic profile of the mesocorticolimbic dopaminergic system.

DA1 receptor mediated regulation of Na+-H+ antiport activity in rat renal cortical brush border membrane vesicles

Our previous studies indicate that dopamine (DA) plays an important role in regulating renal sodium (Na+) metabolism during high Na+ intake, and that DA1 receptors are involved in natriuretic response to acute volume expansion. It has also been shown that in addition to the changes in renal hemodynamics, the natriuretic response produced by exogenously administered DA and DA1 receptor agonists appears to be due to alterations in renal tubular sodium transport mechanisms. This study was designed to investigate the DA1 receptor-mediated changes in Na(+)-H+ antiport activity in tubular brush border membranes of rat kidney. The Na(+)-H+ antiport activity, measured as the amiloride-sensitive Na+ influx in BBMV, was inhibited by 37%, 46%, 33%, and 42% by 1 microM DA, SKF 82958, SKF 38393, and fenoldopam respectively. The DA1 antagonist SCH 23390 increased the antiport activity when given alone, while when administered with an agonist it attenuated the effects of the agonist on the antiporter. DA2 agonists and antagonists failed to affect the antiport activity. These results indicate that the inhibitory effects of DA and DA receptor agonists on Na(+)-H+ antiport activity in renal cortical BBMV were mediated by the DA1 receptors.

Involvement of central dopamine receptors in the hypotensive action of pergolide.

SummaryThe involvement of central dopamine receptors in the hypotensive action of the dopaminergic ergoline, pergolide was determined in anesthetized rats. Intravenous (i.v.) or intracerebroventricular (i.v.t.) administration of pergolide (12.5, 25 and 50 μg/kg) produced dose-dependent decreases in blood pressure. The magnitude of hypotension seen following either i.v. or i.v.t. administration of pergolide was similar. However, while both sulpiride (1 mg/kg, i.v.) as well as phentolamine (1 mg/kg, i.v.) antagonized the hypotensive action of i.v. pergolide, only sulpiride (1 mg/kg, i.v.t.) was able to antagonize the hypotension seen following i.v.t. administration of pergolide. Phentolamine (1 mg/kg, i.v.t.) did not alter the central hypotensive action of pergolide. In a separate group of rats, clonidine (25 μg/kg, i.v.t.) also produced a decrease in blood pressure. While phentolamine (i.v.t.) antagonized the central hypotensive action of clonidine, sulpiride (i.v.t.) did not have any effect on the action of clonidine. These results show that selective activation of central dopamine receptors was responsible for the hypotensive action of centrally-administered pergolide.In a separate group of rats greater splanchnic sympathetic nerve activity was measured. Intravenous pergolide produced similar hypotensive response as seen in previous groups, and this was accompanied by a concomitant decrease in the sympathetic nerve activity. The maximum fall in blood pressure (26±6 mm Hg) was correlated with a 40% reduction in sympathetic nerve activity. The return of blood pressure to control levels occurred after 60–70 min and was also associated with the return of sympathetic nerve activity to control levels. In some experiments administration of sulpiride (i.v.) at a time when maximum falls in blood pressure and sympathetic nerve activity had occurred, caused rapid restoration of both these parameters to control levels.These results provide evidence for the presence of central dopamine receptors in the cardiovascular centers within the brain and suggest that activation of these receptors causes a lowering of blood pressure. Central dopaminergic inhibition of peripheral sympathetic nerve activity may also contribute to the hypotensive action of pergolide.

Region - specific alterations in tyrosine hydroxylase activity in rats exposed to lead

Previous studies from our laboratory showed that subchronic exposure to low levels of Pb resulted in significant decrease in dopamine (DA) content, attenuation of stimulus-induced release of DA in the dopaminergic projection area of nucleus accumbens (NA), and alterations in tyrosine hydroxylase (TH) activity in rat whole brain homogenates. The present study reported here was conducted to assess the functional integrity of DA synthesis in different brain regions of rats subchronically (90-days) exposed to 50 ppm Pb by measuring the activity of the rate limiting enzyme, tyrosine hydroxylase, in seven brain regions. In Pb-exposed rats, TH activity was reduced in two of the seven brain regions investigated, i.e., nucleus accumbens (42% reduction) and frontal cortex (61% reduction) when compared to controls. In contrast, Pb exposure did not affect the TH activity in cerebellum, brainstem, hippocampus, hypothalamus and striatum. The changes in TH activity in nucleus accumbens (NA) and frontal cortex (FC) in Pb-exposed rats were further confirmed by Western blot analysis using TH polyclonal antibody. Collectively, these results indicate that low level subchronic Pb exposure may affect TH protein in these brain regions.