Polychlorinated biphenyl congeners that increase the glucuronidation and biliary excretion of thyroxine are distinct from the congeners that enhance the serum disappearance of thyroxine.

Polychlorinated biphenyl (PCB) congeners differentially reduce serum thyroxine (T(4)) in rats, but little is known about their ability to affect biliary excretion of T(4). Thus, male Sprague-Dawley rats were orally administered Aroclor-1254, Aroclor-1242 (32 mg/kg per day), PCB-95, PCB-99, PCB-118 (16 mg/kg per day), PCB-126 (40 µg/kg per day), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (3.9 µg/kg per day), or corn oil for 7 days. Twenty-four hours after the last dose, [(125)I]T(4) was administered intravenously, and blood, bile, and urine samples were collected for quantifying [(125)I]T(4) and in bile [(125)I]T(4) metabolites. Serum T(4) concentrations were reduced by all treatments, but dramatic reductions occurred in response to Aroclor-1254, PCB-99 [phenobarbital (PB)-type congener], and PCB-118 (mixed-type congener). None of the treatments increased urinary excretion of [(125)I]T(4). Aroclor-1254, PCB-118, TCDD, and PCB-126 (TCDD-type congener) increased biliary excretion of T(4)-glucuronide by 850, 756, 710, and 573%, respectively, corresponding to marked induction of hepatic UDP-glucuronosyltransferase (UGT) activity toward T(4). PCB-95 and PCB-99 did not induce UGT activity; therefore, the increased biliary excretion of T(4)-glucuronide was related to the affinity of congeners for the aryl hydrocarbon receptor. The disappearance of [(125)I]T(4) from serum was rapid (within 15-min) and was increased by Aroclor-1254, PCB-99 and PCB-118. Thus, reductions in serum T(4) in response to PCBs did not always correspond with UGT activity toward T(4) or with increased biliary excretion of T(4)-glucuronide. The rapid disappearance of [(125)I]T(4) from the serum of rats treated with PB-like PCBs suggests that increased tissue uptake of T(4) is an additional mechanism by which PCBs may reduce serum T(4).

Trimethyltin-induced alterations in behavior are linked to changes in PSA-NCAM expression.

The neurotoxic heavy metal trimethyltin (TMT) primarily damages neurons of the hippocampus and limbic areas of the temporal lobe, and causes a dose-dependent decrease in the polysialated form of the neural cell adhesion molecule (PSA-NCAM) in the mouse hippocampus. In the current study, we attempted to associate deficits in spatial learning following TMT exposure at various stages in learning with changes in levels of NCAM-180 and PSA-NCAM in both the hippocampus and frontal cortex. Mice were treated with TMT either before or after training on a spatial learning paradigm and examined for changes in NCAM and PSA-NCAM 12h later. In the first set of experiments, male BALB/c mice were injected with TMT (2.25 mg/kg) or saline i.p. and tested 24-168 h later using hidden and visible versions of the water maze, as well as light avoidance and motor activity. Mice in both treated and control groups which demonstrated a significant improvement in water maze performance also showed an elevation in hippocampal PSA-NCAM at all time points examined. TMT exposure impaired spatial learning and blocked learning-induced elevations in PSA-NCAM expression 24-96 h post-treatment, but these deficits disappeared by 168 h post-treatment. Mice exposed to TMT during reconsolidation of spatial learning (after repeated water maze training) demonstrated a mild and transient difference in escape latency compared to saline exposed mice. TMT administration during this period did not result in the attenuation of PSA-NCAM expression observed when animals were exposed before training. These results confirm a specific role for PSA-NCAM in acquisition and consolidation of spatial memory.

Methylmercury alters Eph and ephrin expression during neuronal differentiation of P19 embryonal carcinoma cells.

Developmental exposure to methylmercury (MeHg) induces a spectrum of neurological impairment characterized by cognitive disturbance, sensory/motor deficit, and diffuse structural abnormalities of the brain. These alterations may arise from neural path-finding errors during brain development, resulting from disturbances in the function of morphoregulatory guidance molecules. The Eph family of tyrosine kinase receptors and their ligands, the ephrins, guide neuronal migration and neurite pathfinding mainly via repulsive intercellular interactions. The present study examined the effects of MeHg on mRNA and protein expression profiles of Ephs and ephrins in the P19 embryonal carcinoma (EC) cell line and its neuronal derivatives. Undifferentiated control P19 cells displayed low- to undetectable levels of mRNA for ephrins or Ephs, with the sole exception of EphA2 which was highly expressed. Upon differentiation into neurons, the ephrin expression increased progressively through day 10. Similarly, expression of the Ephs, including EphsA3, -A4, -A8, -B2, -B3, -B4, and -B6, increased significantly. In contrast, EphA2 expression decreased in day 2, 6 and 10 control neurons. Treatment with MeHg did not affect the expression of mRNA for ephrins or Ephs in undifferentiated P19 cells. However, treatment of differentiating neurons with MeHg for 24 h caused consistent increases in ligand mRNA expression, particularly ephrin-A5, -A6, -B1, and -B2. Similarly, MeHg induced variable increases in mRNA expression of receptors EphA2, -A3, -B3, and -B6. A trend toward a concentration-response relationship was observed for the alterations in Eph receptor mRNA expression although increases at the low and mid concentrations did not reach statistical significance. Immunoblots for ligand and receptor proteins mirrored the increases in the mRNA levels at the 0.5 and 1.5 microM MeHg concentrations but showed decreased protein levels compared to controls at the 3.0 microM concentration. Alterations in the Eph/ephrin family of repulsion molecules may represent an important mechanism in developmental MeHg neurotoxicity.

The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone.

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

Delayed expression of the NFH subunit in differentiating P19 cells.

Pluripotent embryonal carcinoma (P19) cells differentiate into a neural phenotype in response to retinoic acid (RA). Expression of the low and medium molecular weight neurofilament subunits, but not the high molecular weight subunit (NFH), has been reported following RA treatment. In this study NFH expression was detected by Western blotting and immunofluorescence microscopy, but lagged behind the expression of the other subunits in a manner similar to that reported during in vivo neuronal development.

Development of cutaneous amelanotic melanoma in the absence of a functional tyrosinase.

Lack of characteristic pigmentation and a wide range of clinical presentations account for the diagnostic challenge associated with amelanotic malignant melanoma. Experimental studies of this important human cancer have been hampered by the lack of an appropriate animal model. We previously described a transgenic mouse line (TG-3) that spontaneously develops pigmented cutaneous melanoma. F1 crosses were generated with TG-3 and several albino strains, and backcrosses were then made with the albinos. In the present report, we describe the restricted development and characterization of cutaneous amelanotic melanoma in these albino transgenic backcrosses. The incidence and behavior of melanoma in these mice were monitored. A high incidence (80-100%) of spontaneous amelanotic melanoma was observed in albino transgenic mice derived from backcrosses with A, AKR, FVB, and SJL strains. The lowest incidence (30%) was obtained in BALB/c-derived crosses. No tumors were observed in non-transgenic mice. Immunohistochemical and western blot analyses using antibodies against three melanocyte-specific markers of the tyrosinase family of proteins confirmed that the tumors were composed of amelanotic melanocytes. Furthermore, the presence of numerous premelanosomes observed by electron microscopy further supported the melanocytic origin of these tumors. Previous in vitro studies on human melanoma have suggested that cutaneous amelanotic melanoma was evolving from preexisting pigmented cutaneous melanoma. However, our results indicate that it can occur directly, as evidenced by the appearance of cutaneous amelanotic melanoma in the tyrosinase-deficient albino mice. These mice represent a potentially valuable model for studying the mechanistic, diagnostic, and therapeutic features of this highly malignant neoplasm.

Development of early melanocytic lesions in transgenic mice predisposed to melanoma.

We have previously described a line of transgenic mice (TG3) that spontaneously develops heritable malignant melanoma. Histological analysis of these animals during the first postnatal month is described here. In the TG3 line, the number of melanocytes is increased at all anatomical sites to which neural-crest-derived melanocytes normally migrate. Clonal expansion and morphological changes of these melanocytes can be detected as early as postnatal day (PND) 15. By PND 30, cells morphologically indistinguishable from the tumor cells of adult transgenic mice were detected in the ear, eye lid and perianal region. These cells are believed to be the precursors of the primary tumors in adult mice. The stepwise development of melanoma in the TG3 line is similar to the stepwise development of melanoma in humans.

Preferential destruction of cerebellar Purkinje cells by OX7-saporin.

Saporin, a plant toxin derived from Saponaria officinalis, disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex. OX7 is a mouse monoclonal antibody directed against the Thy-1.1 receptor that is differentially expressed on subpopulations of central nervous system neurons. Disulfide conjugation of OX7 to saporin permits delivery of saporin to target neurons. OX7-saporin was used to study the behavioral and morphological consequences of selective destruction of cerebellar Purkinje cells which abundantly express the Thy-1.1 antigen. Male Sprague-Dawley rats received bilateral intraventricular injections of 1- or 2 microg OX7-saporin or 8 microl artificial CSF. Rats were tested for behavioral changes 1 week before and 1, 2, and 8 weeks post-treatment. OX7-saporin treatment resulted in dose- and time-dependent changes in motor performance, activity, and negative geotaxis, but did not affect foot splay. Following behavioral testing, cerebellar sections were prepared for microscopic examination and the pattern of Purkinje cell loss was determined in anatomically matched sections. OX7-saporin induced dose-dependent death of Purkinje cells, particularly in the anterior and superior portions of cerebellar folia 1-6 and folium 9. Other brain regions appeared largely unaffected. Data suggest that intraventricular injection of rats with OX7-saporin is an effective model with which to examine the consequences of Purkinje cell destruction.

Developmental lead exposure disturbs expression of synaptic neural cell adhesion molecules in herring gull brains.

Neurobehavioral testing of herring gull chicks (Larus argentatus) in both laboratory and field studies indicates that lead exposure during critical periods of development causes neurological deficits that may compromise survival in the wild. Accumulating evidence suggests that lead impairs neurodevelopment, in part, by altering the expression of cell adhesion molecules (CAMs) responsible for the proper formation and maintenance of neural structure and synaptic function. We examined the adhesion molecules NCAM, L1, and N-cadherin in gull brains to determine whether these CAMs are altered by lead exposure and might serve as markers of developmental neurotoxicity. One-day-old chicks were collected from nesting colonies and were laboratory housed. On post-hatching day (PHD) 2, chicks were given 100 mg/kg lead acetate or saline (intraperitoneally). Birds were killed on PHD 34, 44, or 55 (blood-lead levels averaged 27.4, 20.8, and 19.5 microg/dl, respectively). Brains were removed and stored at -70 degrees C until analysis. Expression of CAMs was determined in synaptosomal preparations by Western blotting and the activity of NCAM-associated sialyltransferase (ST) was determined in purified whole brain golgi apparatus. Elevation in synaptosomal polysialylated NCAM expression and a significant increase in golgi ST activity was observed in lead-treated animals at PHD 34. Reductions in synaptosomal N-cadherin were observed at PHD 34 and 44, while L1 expression appeared unaffected by lead at any time-point. By 55 days post-hatching, no differences in N-cadherin expression, polysialylated NCAM expression or NCAM-associated ST activity were seen in lead-treated animals as compared with age-matched control animals. Lead-induced disruption of CAM expression during early neurodevelopment may contribute to behavioral deficits observed in herring gulls in both the laboratory and the field, and may serve as a marker for heavy metal exposure during postnatal development.

Mechanisms of injury in the central nervous system.

Neurotoxicants with similar structural features or common mechanisms of chemical action frequently produce widely divergent neuropathologic outcomes. Methylmercury (MeHg) produces marked cerebellar dysmorphogenesis during critical periods of development. The pathologic picture is characterized by complete architectural disruption of neuronal elements within the cerebellum. MeHg binds strongly to protein and soluble sulphydryl groups. Binding to microtubular -SH groups results in catastrophic depolymerization of immature tyrosinated microtubules. However, more mature acetylated microtubules are resistant to MeHg-induced depolymerization. In contrast to MeHg, the structurally similar organotin trimethyltin (TMT) elicits specific apoptotic destruction of pyramidal neurons in the CA3 region of the hippocampus and in other limbic structures. Expression of the phylogenetically conserved protein stannin is required for development of TMT-induced lesions. Inhibition of expression using antisense oligonucleotides against stannin protects neurons from the effects of TMT, suggesting that this protein is required for expression of neurotoxicity. However, expression of stannin alone is insufficient for induction of apoptotic pathways in neuronal populations. The aromatic nitrocompound 1,3-dinitrobenzene (DNB) has 2 independent nitro groups that can redox cycle in the presence of molecular oxygen. Despite its ability to deplete neural glutathione stores, DNB produces edematous gliovascular lesions in the brain stem of rats. Glial cells are susceptible despite high concentrations of reduced glutathione compared with neuronal somata in the central nervous system (CNS). The severity of lesions produced by DNB is modulated by the activity of neurons in the affected pathways. The inherent discrepancy between susceptibility of neuronal and glial cell populations is likely mediated by differential control of the mitochondrial permeability transition in astrocytes and neurons. Lessons learned in the mechanistic investigation of neurotoxicants suggest caution in the evaluation and interpretation of structure-activity relationships, eg, TMT, MeHg, and DNB all induce oxidative stress, whereas TMT and triethyltin produce neuronal damage and myelin edema, respectively. The precise CNS molecular targets of cell-specific lipophilic neurotoxicants remain to be determined.

Modulation of chemokine expression during ischemia/reperfusion in transgenic mice overproducing human glutathione peroxidases.

Renal ischemia/reperfusion (I/R) injury is a major cause of kidney damage. There is accumulating evidence that inflammatory reactions are involved in the pathogenesis of this process. Our studies demonstrate that transgenic mice overexpressing human extracellular and intracellular glutathione peroxidases (GP) are protected against kidney I/R injury. Importantly, significant reduction in neutrophil migration was observed in GP mice compared with nontransgenic mice. Analysis of signaling molecules mediating neutrophil activation and recruitment indicates reduction in the level of KC and macrophage inflammatory protein-2 chemokine expression in transgenic animals. The molecular mechanism mediating this effect appears to involve repression of NF-kappaB activation at the level of IkappaBalpha and IkappaBbeta degradation. In the case of IkappaBalpha, no apparent phosphorylation was detected. These results suggest that IkappaBalpha proteolysis is triggered during the renal I/R pro-oxidant state by a still unknown mechanism, which might be different from other stimuli. A central role of NF-kappaB in CXC chemokine activation was demonstrated in cell culture anoxia/ATP repletion experiments as a model of I/R. The data presented indicate the important role of GP-sensitive signal transduction pathways in the development of inflammatory response and tissue injury during I/R.

Developmental methylmercury administration alters cerebellar PSA-NCAM expression and Golgi sialyltransferase activity.

Brain dysmorphogenesis and persistent psychomotor disturbances are hallmarks of developmental methylmercury (MeHg) exposure, but the molecular mechanisms underlying these effects are poorly understood. Targets of developmental MeHg exposure include neural cell adhesion molecules (NCAMs), sialoglycoconjugate molecules whose proper temporal and spatial expression is important at all stages of neurodevelopment and especially during synaptic structuring. To investigate the effects of MeHg on the temporal expression of NCAM during development, rat pups were dosed with 7.0 mg/kg MeHgCl (s.c.) on alternate days from postnatal days (PNDs) 3-13 and killed on PNDs 15, 30 and 60. Brain MeHg concentrations were determined in a subset of litters injected with CH(3)203Hg. Expression of NCAM180 protein and of NCAM180 polysialylation was examined in whole cerebellum homogenates, cerebellar synaptosomes and isolated cerebellar growth cones by Western blotting and immunocytochemical staining. NCAM sialyltransferase activity was assayed in preparations of purified Golgi apparatus from the cerebelli of rats treated in vivo, or following in vitro incubation with 0, 1, 2.5, or 7.5 microM MeHg for 2 h. At PND15, no change in NCAM180 protein expression was observed in any cerebellar preparations, but decreased polysialylation of NCAM180 was observed in cerebellar whole homogenates, synaptosomes and isolated growth cones. At PND30, both NCAM180 protein expression and NCAM180 polysialylation were elevated in whole homogenate preparations but not in synaptosomes. NCAM180 expression in MeHg-treated rats was similar to controls at PND60, 47 days after the last methylmercury administration. In vivo studies of cerebellar Golgi sialyltransferase activity revealed significant reductions in PND15 MeHg-treated rats as compared to controls, but no changes in sialyltransferase activity in PND30 and PND60 animals. In vitro experiments revealed decreasing sensitivity of cerebellar sialyltransferases to MeHg as the developmental age of the rat increased. Toxic perturbation of the developmentally-regulated expression of polysialylated NCAM during brain formation may disturb the stereotypic formation of neuronal contacts and could contribute to the behavioral and morphological disturbances observed following MeHg poisoning.

Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase.

Acetaminophen is one of the most extensively used analgesics/antipyretics worldwide, and overdose or idiopathic reaction causes major morbidity and mortality in its victims. Research into the mechanisms of toxicity and possible therapeutic intervention is therefore essential. In this study, the response of transgenic mice overexpressing human antioxidant enzymes to acute acetaminophen overdose was investigated. Animals overexpressing superoxide dismutase or plasma glutathione peroxidase demonstrated dramatic resistance to acetaminophen toxicity. Intravenous injection of glutathione peroxidase provided normal mice with nearly complete protection against a lethal dose of acetaminophen. Surprisingly, animals overexpressing intracellular glutathione peroxidase in the liver were significantly more sensitive to acetaminophen toxicity compared with nontransgenic littermates. This sensitivity appears to be due to the inability of these animals to efficiently recover glutathione depleted as a result of acetaminophen metabolism. Finally, the results suggest that glutathione peroxidase overexpression modulates the synthesis of several acetaminophen metabolites. Our results demonstrate the ability of glutathione peroxidase levels to influence the outcome of acetaminophen toxicity.

Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage.

As stroke is a major cause of disability and death in the western world, there is great interest in the basic mechanisms by which ischemia/reperfusion (I/R) causes damage. To this end, extensive research has been carried out which identifies reactive oxygen species (ROS) as key participants in brain damage resultant from I/R. Brain tissue is protected from ROS damage by antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GP). Overexpression of SOD in transgenic mice has already been demonstrated to confer protection against I/R damage in murine stroke models. We are using transgenic mice overexpressing the intracellular form of glutathione peroxidase (GP1) to determine the protective capacity of overexpression of this enzyme on stroke damage. 1 h of focal cerebral ischemia followed by 24 h of reperfusion was induced using the intraliminal suture method. Volume of infarction was reduced by 48% in GP1 mice compared to nontransgenic littermates. Brain edema was reduced by 33%. Behavioral deficits agreed with histologic data. Overexpression of glutathione peroxidase confers significant protection against I/R damage in our stroke model possibly through direct scavenging of ROS or through the influencing of signalling mechanisms which lead to tissue damage.

Development of heritable melanoma in transgenic mice.

Transfer of genetic material into recipient cells by transfection has been used successfully to isolate genes responsible for particular phenotypic traits. By using this strategy, DNA fragments were isolated that when transfected into appropriate uncommitted cells will commit the recipient cells to undergo adipocyte differentiation. Transgenic mice were generated with one of the active DNA clones, Clone B. The transgenic mice were expected to display an adipocyte-related phenotype; however, the animals developed melanin containing tumors at a young age. Insertion of Clone B into the mouse DNA probably interrupted a gene(s) that is involved in the regulation of cell growth, specifically regulation of cell growth in melanin-producing cells. Histopathologic analysis of these mice showed dark spots on the ear lobes of the animals as early as 10-12 d of age. By 3 mo, in addition to the ear lobes, pigmented tumors could be observed in other organs. A significant number of these transgenic mice died within 1 y of age. The melanomas developed spontaneously in these animals in the absence of any known chemical carcinogen or ultraviolet radiation. This line of mice provides a way of identifying genes involved in regulation of cell growth control and differentiation. These mice also serve as a model system to investigate the molecular, genetic, and phenotypic characterization and development of melanomas.

Development of esophageal metaplasia and adenocarcinoma in a rat surgical model without the use of a carcinogen.

In order to establish an animal model for studying the cause and prevention of esophageal adenocarcinoma (EAC) and its frequent precursor, Barrett's esophagus (BE), factors affecting the pathogenic processes were investigated in an esophagoduodenal anastomosis model with rats. Experiments by us and others have shown that surgical treatment produced reflux esophagitis with cell hyperproliferation, but not EAC. Additional treatment with a carcinogen has been shown to be necessary for the development of EAC, squamous cell carcinomas (SCC) or EAC/SCC mixtures. We found that the surgically treated animals developed anemia due possibly to reduced iron absorption. When the operated animals were supplemented with iron, EAC occurred at a high rate (73%) after 30 weeks, and treatment with N'-nitrosonornicotine did not enhance the rate of tumorigenesis. Treatment with carcinogen, however, induced SCC in the group of rats killed after 22 weeks. The results suggest that iron overload, which is known to cause oxidative damage, is an enhancing factor for adenocarcinogenesis. The pathogenesis of EAC in the iron-supplemented, non-carcinogen treated group resembles human esophageal adenocarcinogenesis in many features. All the BE was the specialized type with goblet cells (containing sialomucin or sulfomucin) and columnar cells (containing acid or neutral mucin) as well as an incompletely developed brush border. Almost all of the BE was located at the bottom of the esophagus and was continuous with the duodenal mucosa; dysplasia became more frequent at later time points. All of the cancers were well-differentiated mucinous EAC, and most of the EAC had an adjacent area of BE with dysplasia. The results are consistent with the proposed human sequence for pathogenic events of BE progression to 'BE with dysplasia' and then to EAC. Esophagoduodenal anastomosis and iron treatment in rats produces a high rate of BE and EAC which are morphologically similar to human BE and EAC; this may be a useful animal model to study the development and prevention of EAC in humans.

Altered sensitivity of posttranslationally modified microtubules to methylmercury in differentiating embryonal carcinoma-derived neurons.

The effects of methylmercury (MeHg) on microtubules (MTs) in differentiating neurons derived from retinoic acid-induced embryonal carcinoma (EC) cells in culture were examined by immunofluorescence microscopy. Undifferentiated EC cells contained mostly kinetically labile tyrosinated (TYR) MTs which extended from the centrosome and a small population of stable acetylated (ACT) MTs usually (but not exclusively) associated with the centrosome. TYR MTs of undifferentiated cells underwent concentration- and time-dependent disassembly upon exposure to low concentrations of MeHg (1-2 microM), whereas ACT MTs were resistant to MeHg at low concentrations, with many remaining intact even in 5 microM MeHg. After 2 days in retinoic acid the appearance of short neuritic processes was indicative of early differentiation. TYR MTs predominated both in the short neurites and cell soma and remained susceptible to low concentrations of MeHg. ACT MTs also extended into the short neurites but were most often found in small bundles within the perikarya. ACT MTs remained more resistant to MeHg than TYR MTs. The neuron-specific tubulin isotype betaIII was first detected during the second day of differentiation. MTs labeled with antibodies to betaIII showed similar sensitivity to MeHg as TYR MTs, suggesting that MTs containing betaIII were largely tyrosinated. After 4 and 6 days of differentiation a greater number of betaIII-positive neurons were present with progressively longer and often branching neurites. TYR MTs were present in cell soma and neurites while ACT MTs were found almost exclusively in neurites. TYR MTs remained highly susceptible to MeHg (most notably in perikarya) in comparison to ACT MTs at all stages of differentiation; however, with increasing time in culture, even TYR MTs gained appreciable stability to MeHg. These data indicate that microtubules in developing neurons become progressively more resistant to disassembly by MeHg, suggesting that the most critical period of MT susceptibility occurs very early in development in vivo.

Effects of phenethyl isothiocyanate on acetaminophen metabolism and hepatotoxicity in mice.

Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous and other vegetables, is a potent inhibitor of cytochrome P450 2E1. This enzyme catalyzes the bioactivation of acetaminophen (APAP) and many other xenobiotics. The present study investigated the effects of PEITC on APAP metabolism and associated hepatotoxicity in Swiss-Webster mice. When PEITC (19-150 micromol/kg) was given to mice intragastrically 1 hr before or immediately prior to a toxic dose of APAP, the APAP-induced hepatotoxicity was significantly decreased or was completely prevented. The extent of toxicity was evaluated by mortality, serum levels of glutamic-pyruvic transaminase, lactate dehydrogenase, and liver histopathology. Pretreatment of mice with ethanol enhanced APAP hepatotoxicity; this enhanced toxicity could also be prevented by the administration of PEITC. PEITC treatment prevented the depletion of hepatic glutathione levels caused by oxidized APAP metabolites. PEITC treatment also significantly decreased the plasma levels of oxidized APAP metabolites (analyzed as APAP-glutathione, APAP-cysteine, and APAP-N-acetylcysteine) and reduced the urinary excretion of APAP-cysteine. In microsomal incubations, PEITC effectively inhibited the rate of APAP-glutathione formation from APAP as well as the P450 2E1-dependent N-nitrosodimethylamine demethylase and the P450 1A2-dependent ethoxyresorufin O-deethylase activities. The protective action of PEITC against APAP toxicity is attributed to the blocking of APAP activation through inhibition of P450 enzymes.

Altered expression of polysialylated NCAM in mouse hippocampus following trimethyltin administration.

Proper structuring of neural connections in the hippocampus is mediated by cell adhesion molecules, membrane-linked proteins involved in cell recognition and stabilization of cytoarchitecture. Modulated expression of the neural cell adhesion molecule (NCAM) at the synapse permits plasticity required for both learning and memory. Polysialylation of NCAM, particularly the synapse-specific 180 kDa isoform (NCAM180), allows hippocampal neurons to alter their neuronal connections during learning acquisition and memory consolidation in mature brain. These activity-dependent changes in NCAM expression represent a sensitive target for neurotoxicity. Trimethyltin (TMT), a potent hippocampal neurotoxicant, alters total NCAM expression in whole mouse hippocampus and impairs learning in rodents. To investigate the expression of polysialylated NCAM following TMT administration, Swiss-Webster mice were injected (i.p.) with 2.0 or 3.0 mg TMT/kg and sacrificed 6 hrs to 7 days later. Immunocytochemical staining for polysialylated NCAM (PSA-NCAM) revealed marked reduction of staining of hippocampal dentate granule cells 6-72 hours after TMT treatment. Partial recovery of hippocampal polysialylated NCAM was observed after 7 days. Immunoblot data indicated that loss of PSA-NCAM expression paralleled reductions seen in NCAM180 and markers of cytoskeletal integrity. Assays for proteolytic activity in hippocampus revealed rapid, reversible protease activation which correlated temporally with the reduction of NCAM180 and PSA-NCAM. Proteolytic degradation following hippocampal injury may serve to disrupt NCAM-mediated adhesion. Protracted loss of polysialylated NCAM in dentate gyrus following injury may serve as a useful marker in toxicant-induced learning disorders.

Perinatal methanol exposure in the rat. I. Blood methanol concentration and neural cell adhesion molecules.

Although the acute toxicity of methanol is well documented, few studies have addressed the consequences of perinatal exposures to the low concentrations that are expected to arise from its proposed use as a component of automobile fuel. This report describes the general research design of a series of studies, the effects of methanol exposures on blood concentrations in dams and neonates, and indices of brain development. Four cohorts of Long-Evans pregnant rats, each cohort consisting of an exposure (n = 12) and a control (n = 12) group, were exposed whole-body to 4500 ppm methanol vapor or air for 6 hr daily beginning on Gestation Day 6. Both dams and pups were then exposed through Postnatal Day 21 (PND 21). Blood methanol concentrations determined by gas chromatography from samples obtained immediately following a 6-hr exposure reached approximately 500-800 micrograms/ml in the dams during gestation and lactation. Average concentrations for pups attained levels about twice those of the dams. Selected offspring from Cohort 4 were exposed for one additional 6-hr session at ages that extended out to PND 52. Regression analyses showed that the blood methanol concentrations of the pups declined until about PND 48, at which time their levels approximated those of their dams. Such pharmacokinetic differences might increase the risks posed to developing organisms. Light-microscopic analysis showed no significant abnormalities in the brains of the methanol-treated animals. However, assays of neural cell adhesion molecules (NCAMs) in brains of pups sacrificed on PND 4 showed staining for both the 140 and the 180 kDa isoforms to be less intense in the cerebellum of exposed animals. NCAM differences were not apparent in animals sacrificed 15 months after their final exposure.