Biomedical subjects

M P Waalkes

Publications and source records attributed to M P Waalkes.

At least 19 recordsLinked to original sources

PubMed2001

Genetic background but not metallothionein phenotype dictates sensitivity to cadmium-induced testicular injury in mice.

Sensitivity to cadmium (Cd)-induced testicular injury varies greatly among mouse strains. For instance, 129/SvJ (129) mice are highly sensitive while C57BL/6J (C57) mice are refractory to Cd-induced testicular injury. Metallothionein (MT), a Cd-binding protein, is thought to be responsible for the strain susceptibility to Cd toxicity. In this study, MT-I/II knockout (MT-null) and wild-type 129 mice were used to determine the role of MT in Cd-induced testicular injury. Two additional strains of mice (C57 and the C57 x 129 F1cross) were also used to help define the role of genetic background in Cd toxicity. Mice were given 5-20 micromol/kg ip CdCl(2) and testicular injury was examined 24 h later by histopathology and testicular hemoglobin concentration. Cd produced dose-dependent testicular injury in all strains of mice, except for C57 mice, in which testicular injury could not be produced. MT-null mice were more sensitive than C57 x 129 mice but were equally sensitive as 129 mice to Cd-induced testicular injury. Fourteen days after 15 micromol/kg ip Cd administration, testicular atrophy was evident in MT-null, 129, and C57 x 129 mice but was absent in C57 mice. The resistance of C57 mice to Cd-induced testicular injury could not be attributed solely to a decreased uptake of (109)Cd nor to a greater amount of testicular MT. Microarray analysis revealed a higher expression of glutathione peroxidase in the testes of C57 mice, as well as genes encoding antioxidant components and DNA damage/repair, but their significance to Cd-induced injury is not immediately clear. Thus, this study demonstrates that it is genetic strain, not MT genotype, that is mechanistically important in determining susceptibility to Cd-induced testicular injury.

Animals↗

PubMed2001

Association of c-myc overexpression and hyperproliferation with arsenite-induced malignant transformation.

Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the liver. However, inorganic arsenic has never been unequivocally shown to be an animal carcinogen, and its carcinogenic mechanism remains undefined. Our previous studies indicate that chronic (> or =18 weeks), low-level (125 to 500 nM) exposure to arsenite induces malignant transformation in the normally nontumorigenic rat liver epithelial cell line (TRL 1215), and these chronic arsenic-exposed (CAsE) cells produce invasive and metastatic tumors upon inoculation into nude mice. In addition, a prior microarray screening analysis of aberrant gene expression showed several oncogenes were overexpressed in CAsE cells exposed to 500 nM arsenite, including a prominent overexpression of the protooncogene c-myc, as well as genes related to cell proliferation. Thus, to better understand the mechanism of arsenic carcinogenesis, we studied the role of c-myc overexpression in arsenite-induced cell transformation. The upregulation of c-myc was confirmed by RT-PCR at the transcription level and by Western blot analysis for the translation product. Further analysis showed that arsenite produced significant increases in the steady-state expression of c-myc in a time- and concentration-dependent manner during the malignant transformation process. The level of c-myc expression was highly correlated (r = 0.988) with tumor formation after inoculation of CAsE cells into nude mice and was also highly correlated (r = 0.997) with genomic DNA hypomethylation. CAsE cells showed a high cell proliferation rate in a fashion related to the level of arsenic exposure. The expression of c-myc was highly correlated with cellular hyperproliferation (r = 0.961). Consistent with the enhanced proliferation both proliferating cell nuclear antigen and cyclin D1 were overexpressed in CAsE cells. In summary, a prominent overexpression of c-myc, a gene frequently activated during hepatocarcinogenesis, is strongly correlated with several events possibly associated with arsenic-induced malignant transformation, including hyperproliferation, DNA hypomethylation and tumor formation upon inoculation into nude mice. These correlations provide convincing evidence c-myc overexpression is mechanistically important in arsenic-induced malignant transformation in this model system.

Animals↗

PubMed2001

Cyproterone acetate induces a cellular tolerance to cadmium in rat liver epithelial cells involving reduced cadmium accumulation.

Several reports indicate that some steroids, in particular sex steroid hormones, can modify cadmium toxicity. We recently reported that cyproterone acetate (CA), a synthetic steroidal antiandrogen that is closely related in structure to progesterone, affects cadmium toxicity in mice. In the present study, we investigated the effect of CA on cadmium toxicity in a rat liver epithelial cell line (TRL 1215) in vitro. Cells were exposed to various concentrations of CA (0,1,10, or 50 microM) for 24 h and subsequently exposed to cadmium (0,50, or 100 microM; as CdCl2) for an additional 24 h. CA pretreatment resulted in a clear decrease in the sensitivity to cadmium. Additional time course study showed CA pretreatment provided protection against cadmium toxicity but only when given for 6 or more hours prior to cadmium exposure. Cellular cadmium accumulation was markedly reduced (60% decrease) in cells pretreated for 6 or more hours with CA. In the presence of protein synthesis inhibitors the protective effect of CA toward cadmium toxicity was abolished. However, in the presence of the GSH synthesis inhibitor, L-buthionine (S,R)-sulfoximide (BSO), the protective effect of CA toward cadmium toxicity remained. CA alone increased metallothionein (MT) levels 2.4-fold, while cadmium (50 microM) alone resulted in a 8.9-fold increase over control. However, cadmium-induced MT synthesis was markedly decreased by CA pretreatment probably because of reduced cadmium accumulation. Analysis of various metal transporters by bDNA signal amplification assay revealed that the ZnT-1 transporter gene, which encodes for a membrane protein associated with zinc efflux, was expressed three-fold more in CA treated cells than control. These data show that CA pretreatment provides protection against cadmium toxicity in vitro and indicate that this protection is due to a decreased accumulation of cadmium rather than through activation of MT synthesis. This decrease of cellular cadmium accumulation appears to be related to events that require protein synthesis and may be due to activation of the genes associated with zinc efflux.

Androgen Antagonists↗

PubMed2001

Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

Amino Acid Sequence↗

PubMed2001

Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT.

The metal binding protein metallothionein (MT) is involved in zinc homeostasis since it typically binds large amounts of zinc. Free zinc can control MT gene expression by interacting with metal-sensitive transcription factors. However, the precise factors governing intracellular release of metal ions from MT remain unknown. Aerobic nitric oxide (NO) can nitrosate thiol groups in proteins, and MT-bound cadmium is released by NO exposure. Thus, we hypothesized that NO may also be effective at displacing zinc from MT in cultured cells and that this could be an important physiological control mechanism in zinc homeostasis and utilization. In this study, DETA/NO, an agent that spontaneously generates NO with a 20-h half life in physiological media, was used to study the release of zinc from MT and the induction of MT in TRL1215 cells (a normal rat liver cell line). Zinc or cadmium was given at levels inducing MT production, followed by DETA/NO (20-200 microM) to produce controlled NO exposure in both cell lines. Although both metals activated MT gene expression, MT-I mRNA and MT protein were further increased when DETA/NO was given after zinc or cadmium treatment. Additionally, NO from DETA/NO clearly displaced MT-bound zinc, as evidenced by G-75 gel-filtration chromatography. The released zinc or cadmium probably then stimulates further MT gene expression. These results suggest that NO may play an important role in regulation of cellular zinc homeostasis by providing a controlled release mechanism for metal ions stored in MT, and NO-mediated release of MT-bound zinc could in turn activate gene expression, such as with the MT gene.

Animals↗

PubMed2001

Cadmium-induced malignant transformation of human prostate epithelial cells.

Prostate cancer has become epidemic, and environmental factors such as cadmium may be partly responsible. This study reports malignant transformation of the nontumorigenic human prostatic epithelial cell line RWPE-1 by in vitro cadmium exposure. The cadmium-transformed cells exhibited a loss of contact inhibition in vitro and rapidly formed highly invasive and occasionally metastatic adenocarcinomas upon inoculation into mice. The transformed cells also showed increased secretion of MMP-2 and MMP-9, a phenomenon observed in human prostate tumors and linked to aggressive behavior. Cadmium-induced malignant transformation of human prostate epithelial cells strongly fortifies the evidence for a potential role of cadmium in prostate cancer.

Adenocarcinoma↗

PubMed2001

Genetic events associated with arsenic-induced malignant transformation: applications of cDNA microarray technology.

Arsenic is a human carcinogen. Our recent work showed that chronic (>18 wk), low-level (125-500 nM) arsenite exposure induces malignant transformation in normal rat liver cell line TRL1215. In these arsenic-transformed cells, thecellular S-adenosylmethionine pool was depleted from arsenic metabolism, resulting in global DNA hypomethylation. DNA methylation status in turn may affect the expression of a variety of genes. This study examined the aberrant gene expression associated with arsenic-induced transformation with the use of Atlas Rat cDNA Expression microarrays. Poly(A(+)) RNA was prepared from arsenic-transformed cells and passage-matched control cells, and (32)P-labeled cDNA probes were synthesized with Clontech Rat cDNA Synthesis primers and moloney murine leukemia virus reverse transcriptase. The hybrid intensity was analyzed with AtlasImage software and normalized with the sum of the four housekeeping genes. Four hybridizations from separate cell preparations were performed, and mean and SEM for the expression of each gene were calculated for statistical analysis. Among the 588 genes, approximately 80 genes ( approximately 13%) were aberrantly expressed. These included genes involved in cell-cycle regulation, signal transduction, stress response, apoptosis, cytokine production and growth-factor and hormone-receptor production and various oncogenes. These initial gene expression analyses for the first time showed potentially important aberrant gene expression patterns associated with arsenic-induced malignant transformation and set the stage for numerous further studies. Mol. Carcinog. 30:79-87, 2001. Published 2001 Wiley-Liss, Inc.

Animals↗

PubMed2001

Effect of abdominal surgery on the activity of acid and alkaline ribonucleases in rats.

Ribonucleases (RNases) are a group of enzymes that hydrolyze different classes of RNA. It has been suggested that RNase activity in cells can act to indirectly regulate protein synthesis by controlling RNA degradation. However, little is known about the role of RNases under conditions characterized by a sudden increase of protein synthesis, such as with surgical trauma. The aim of this study was to investigate the effect of abdominal surgery on acid and alkaline RNase activities in rat liver. Acid and alkaline RNase activities decreased significantly at 3 h after surgery, reaching the lowest level at 16 h (63% less than control) for the acid and 6 h (39% less than control) for the alkaline activities. Acid RNase activity returned to its initial values 20 h after surgery, while alkaline RNase activity remained decreased even 24 h after surgery. In order to determine whether the observed decreases in RNase activity were produced by RNase inhibitors (RIs), the enzymatic activities of both RNases were measured after the addition of zinc, to dissociate possible RI/RNase complexes. Zinc addition increased acid RNase activity by 61%, but had no significant effect on alkaline RNase activity. Administration of cycloheximide (an inhibitor of protein synthesis) 2 h before surgery prevented the decrease of acid RNase activity 12 h after surgery, while there was no effect on the decrease in alkaline RNase activity. These results show that surgery produces a decrease in hepatic acid and alkaline RNase activities. The decreased acid RNase activity could be a consequence of the de novo synthesis of RNase inhibitors as a response to surgical trauma, while the mechanism involved in the decrease of alkaline RNase activity is unclear. Under pathophysiological conditions, which induce a high rate of protein synthesis, such as surgical wounding, decreased acid and alkaline RNase activity could provide an important mechanism for enhanced protein synthesis, by prolonging RNA half-life.

Abdomen↗

PubMed2001

Application of cDNA microarray to the study of arsenic-induced liver diseases in the population of Guizhou, China.

Arsenic is an environmental toxicant and a human carcinogen. Epidemiology studies link human arsenic exposure to various diseases and cancers, including liver diseases and hepatocellular carcinoma. However, the molecular mechanisms for arsenic toxicity and carcinogenicity are poorly understood. To better understand these mechanisms, we used the human cancer cDNA expression array to profile aberrant gene expression in arsenic-exposed populations in Guizhou, China. The selected patients had a history of exposure to environmental arsenic for at least 6-10 years, and had arsenic-induced skin lesions and hepatomegaly. Samples were obtained by liver needle biopsy. Histology showed degenerative liver lesions, such as chronic inflammation, vacuolation, and focal necrosis. The University of North Carolina Hospitals provided normal human liver tissues from surgical resection or rejected transplants. Microarray was performed with total RNA from liver samples, and signal intensities were analyzed with AtlasImage software and normalized with 9 housekeeping genes. Means and SEM were calculated for statistical analysis. Approximately 60 genes (10%) were differentially expressed in arsenic-exposed human livers compared to controls. The differentially expressed genes included those involved in cell-cycle regulation, apoptosis, DNA damage response, and intermediate filaments. The observed gene alterations appear to be reflective of hepatic degenerative lesions seen in the arsenic-exposed patients. This array analysis revealed important patterns of aberrant gene expression occurring with arsenic exposure in human livers. Aberrant expressions of several genes were consistent with the results of array analysis of chronic arsenic-exposed mouse livers and chronic arsenic-transformed rat liver cells. Clearly, a variety of gene expression changes may play an integral role in arsenic hepatotoxicity and possibly carcinogenesis.

Arsenic↗

PubMed2001

Stress-related gene expression in mice treated with inorganic arsenicals.

Arsenic (As) is an environmental chemical of high concern for human health. Acute toxicity of arsenic is dependent on its chemical forms and proximity to high local arsenic concentrations is one of the mechanisms for cell death. This study was designed to define acute arsenic-induced stress-related gene expression in vivo. Mice were injected sc with either sodium arsenite [As(III), 100 micromol/kg], sodium arsenate [As(V), 300 micromol/kg], or saline. To examine stress-related gene expression, livers were removed 3 h after arsenic injection for RNA and protein extraction. The Atlas Mouse Stress/Toxicology array revealed that the expression of genes related to stress, DNA damage, and metabolism was altered by acute arsenic treatments. Expression of heme oxygenase 1 (HO-1), a hallmark for arsenic-induced stress, was increased 10-fold, along with increases in heat shock protein-60 (HSP60), DNA damage inducible protein GADD45, and the DNA excision repair protein ERCC1. Downregulation of certain cytochrome P450 enzymes occurred with arsenic treatment. Multiprobe RNase protection assay revealed the activation of the c-Jun/AP-1 transcription complex after arsenic treatments. Western blot analysis further confirmed the enhanced production of arsenic-induced stress proteins such as HO-1, HSP70, HSP90, metallothionein, the metal-responsive transcription factor MTF-1, nuclear factor kappa B and c-Jun/AP-1. Increases in caspase-1 and cytokines such as tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 were also evident. In summary, this study profiled the gene expression pattern in mice treated with inorganic arsenicals, which adds to our understanding of acute arsenic poisoning and toxicity.

Animals↗

PubMed2001

Mechanisms of arsenic-induced cross-tolerance to nickel cytotoxicity, genotoxicity, and apoptosis in rat liver epithelial cells.

The purpose of the present study was to investigate the mechanism of cross-tolerance to nickel in arsenic-transformed cells. Chronic arsenite-exposed (CAsE) cells (TRL 1215 cells, which had been continuously exposed to 0.5 microM arsenite for 20 or more weeks) and control TRL 1215 cells were both exposed to nickel for 24 h, and cell viability was determined by metabolic integrity. The LC(50) for nickel was 608 +/- 32 microM in CAsE cells as compared to 232 +/- 16 microM in control cells, a 2.6-fold increase. CAsE and control cells were treated with 200 microM nickel for 4 h and cellular-free radical production was measured using ESR spectrometry. Hydroxyl radical generation was decreased in CAsE cells. Thiobarbituric acid reactive substances, indicative of lipid peroxidation, and 8-oxo-2'-deoxyguanosine, indicative of oxidative DNA damage, were reduced in CAsE cells. Flow cytometric analysis using Annexin/FITC revealed that nickel-induced apoptosis was reduced in CAsE cells. CAsE cells showed generalized resistance to oxidant-induced toxicity as evidenced by a marked reduction in sensitivity to hydrogen peroxide. Interestingly, intracellular reduced glutathione (GSH) levels were significantly increased in CAsE cells, and when GSH was depleted, CAsE cells lost their nickel resistance. The mechanism of arsenic-induced cross-tolerance to cytotoxicity, genotoxicity, and apoptosis induced by nickel appears related to a generalized resistance to oxidant-induced injury, probably based, at least in part, in increased cellular GSH levels.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Induction of apoptosis in cells by cadmium: quantitative negative correlation between basal or induced metallothionein concentration and apoptotic rate.

Metallothionein (MT) often reduces the adverse effects of cadmium (Cd), but how it may alter Cd-induced apoptosis is unclear. The goal of this study was to define the role of MT in Cd-induced apoptosis using cell lines with widely varying sensitivity to Cd. Effects of Cd on growth of human hepatocellular carcinoma cell lines (HepG2 and PLC/PRF/5) were investigated and compared with Chang cells. These cells were cultured with 0, 5, 10, 20, 40, 80, and 120 microM of Cd for 3, 6, 12, and 24 h. Significant cytolethality was observed in HepG2 and PLC/PRF/5 cells in a time- and concentration-dependent manner, with LC(50) values of 24 microM and 13 microM, respectively. However, Chang cells were much less sensitive to Cd-induced cytotoxicity (LC(50), 64 microM). Apoptotic cell death occurring at cytolethal concentrations was demonstrated in all cell lines by DNA fragmentation on agarose gel electrophoresis or by ELISA. When MT was measured, there was a highly significant negative linear correlation between the basal cellular MT concentration or Cd-induced MT and the rate of apoptosis induced by Cd in these cell lines. Treating HepG2 cells with zinc (Zn) made the relatively sensitive HepG2 cell line resistant to Cd-induced apoptosis, likely due to Zn-induced MT. In fact, there was also a significant negative linear correlation between the amount of Zn-induced MT in HepG2 cells and the rate of Cd-induced apoptosis. These findings revealed that basal or induced MT perturbs Cd-induced apoptotic cell death in various cell lines, and a strong negative correlation exists between cellular MT content and the rate of apoptosis induced by Cd.

Apoptosis↗

PubMed2001

Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic.

Recent work shows that long-term exposure to low levels of arsenite induces malignant transformation in a rat liver epithelial cell line. Importantly, these chronic arsenic-exposed (CAsE) cells also develop self-tolerance to acute arsenic exposure. Tolerance is accompanied by reduced cellular arsenic accumulation, suggesting a mechanistic basis for reduced arsenic sensitivity. The present study examined the role of xenobiotic export pumps in acquired arsenic tolerance. Microarray analysis of CAsE cells showed increased expression of the genes encoding for glutathione S-transferase Pi (GST-Pi), multidrug resistance-associated protein genes (MRP1/MRP2, which encode for the efflux transporter Mrp1/Mrp2) and the multidrug resistance gene (MDR1, which encodes for the efflux transporter P-glycoprotein). These findings were confirmed at the transcription level by reverse transcription-polymerase chain reaction and at the translation level by Western-blot analysis. Acquired arsenic tolerance was abolished when cells were exposed to ethacrynic acid (an inhibitor of GST-Pi), buthionine sulfoximine (a glutathione synthesis inhibitor), MK571 (a specific inhibitor for Mrps), and PSC833 (a specific inhibitor for P-glycoprotein) in dose-dependent fashions. MK571, PSC833, and buthionine sulfoximine markedly increased cellular arsenic accumulation. Consistent with a role for multidrug resistance efflux pumps in arsenic resistance, CAsE cells were found to be cross-resistant to cytotoxicity of several anticancer drugs, such as vinblastine, doxorubicin, actinomycin-D, and cisplatin, that are also substrates for Mrps and P-glycoprotein. Thus, acquired tolerance to arsenic is associated with increased expression GST-Pi, Mrp1/Mrp2 and P-glycoprotein, which function together to reduce cellular arsenic accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Chronic combined exposure to cadmium and arsenic exacerbates nephrotoxicity, particularly in metallothionein-I/II null mice.

Cadmium (Cd) and arsenic (As) are important inorganic toxicants in the environment. Humans certainly have the potential to be exposed to the mixtures of Cd and As, but the toxicological interactions of these inorganic mixtures are poorly defined. Metallothionein (MT) is a cysteine-rich, metal-binding protein that plays an important role in Cd detoxication, but its role in As toxicity is less certain. To examine the role of MT in Cd- and/or As-induced nephrotoxicity, MT-I/II-knockout (MT-null) mice and background-matched wild-type (WT) mice were fed CdCl(2) (100 ppm Cd) in the diet, NaAsO(2) (22.5 ppm As) in the drinking water, or Cd plus As for 4 months. Subsequently, nephrotoxicity was examined by morphological and biochemical techniques. Chronic exposure to Cd produced more renal toxicity than As, and the combination of Cd and As produced even more renal injury than caused by either of the chemicals given alone. In mice receiving Cd plus As, proximal tubule degeneration and atrophy, glomerular swelling and interstitial fibrosis were more severe than those produced by either inorganic. Furthermore, lack of MT rendered MT-null mice more sensitive than WT mice to the nephrotoxicity produced by chronic Cd- and/or As-exposure. MT-null mice were especially susceptible to the toxicity produced by the combination of Cd and As, as evidenced by decreased body weight, enzymuria, glucosuria, proteinuria and nephropathy. In conclusion, this study indicates that As may potentiate Cd nephrotoxicity during the long-term, combined exposure, and that intracellular MT plays a role in decreasing the nephropathy of combined exposure to Cd and As.

Acetylglucosaminidase↗

PubMed2000

Induction of proliferative lesions of the uterus, testes, and liver in swiss mice given repeated injections of sodium arsenate: possible estrogenic mode of action.

Inorganic arsenic (As) is a human carcinogen but has not been unequivocally proven carcinogenic in rodents. For instance, one older study indicates that repeated iv injections of sodium arsenate might induce lymphomas in Swiss mice (58% incidence) (Osswald and Goerttler, Verh. Dtsch. Ges. Pathol. 55, 289-293, 1971), but it was considered inadequate for critical evaluation of carcinogenic potential largely because of issues in experimental design. Therefore, we studied repeated iv sodium arsenate injection and neoplastic response in male and female Swiss mice. Groups (n = 25) of mice received sodium arsenate (0.5 mg/kg, iv) or saline (control) once/week for 20 weeks and were observed for a total of 96 weeks when the study ended. Differences in survival and body weights were unremarkable. In females, arsenate induced marked increases in the incidence and severity of cystic hyperplasia of the uterus compared against controls. Arsenate also was associated with a rare adenocarcinoma of the uterus. Hyperplastic uterine epithelium from arsenate-exposed animals showed strong positive immunostaining for the proliferating cell nuclear antigen (PCNA). There was also an upregulation of estrogen receptor (ER) immunoreactive protein in the early lesions of uterine luminal and glandular hyperplasia, although a progressive decrease in its expression was seen in the severe hyperplastic or neoplastic epithelium. In common with the preneoplastic and neoplastic gynecological lesions in humans, the levels of immunoreactive inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine-containing proteins were greater in the uterine hyperplastic epidermis and their intensity was positively correlated with the severity of the lesions. Arsenate-induced uterine hyperplastic lesions also showed a strong upregulation of cyclin D1, an estrogen-associated gene product essential for progression through the G1 phase of the cell cycle. In other tissues, arsenate increased testicular interstitial cell hyperplasia incidence and severity over control but without affecting the incidence of tubular degeneration. Arsenate also induced increases in hepatic proliferative lesions (HPL; foci of alteration + neoplasia), but only in females. Significant skin changes (incidence of hyperkeratotic lesions) and renal lesions (severity of nephropathy) also occurred in arsenate-treated females. Thus, repeated arsenate exposure, though not outright tumorigenic in the present study, was associated with proliferative, preneoplastic lesions of the uterus, testes, and liver. Estrogen treatment has been associated with proliferative lesions and tumors of the uterus, female liver, and testes in other studies, supporting a hypothesis that arsenate might somehow act through an estrogenic mode of action.

Animals↗

PubMed2000

Cadmium induces c-myc, p53, and c-jun expression in normal human prostate epithelial cells as a prelude to apoptosis.

Cadmium is a suspected human prostatic carcinogen shown to induce prostatic tumors and proliferative lesions in rats. The carcinogenic mechanism of cadmium is unknown, but its poor mutagenicity points toward an epigenetic mechanism. Here we studied the effect of cadmium on genes involved in growth regulation of prostate epithelial cell using the human prostate epithelial cell line RWPE-1, which is immortalized but not transformed and is androgen-responsive. Treatment with 10 microM cadmium resulted in transient increases in c-myc and p53 mRNA levels that peaked at 2-fold and 1.4-fold, respectively, compared to control after 2 h. In contrast, c-jun mRNA levels were increased >3-fold after 2, 4, and 6 h and 20-fold after 24 h. DNA synthesis decreased after 24 h of cadmium exposure. Further study revealed a significant increase in apoptosis after 48 h of cadmium exposure. However, approximately 35% of the cells were still viable and appeared normal, indicating this subpopulation was more resistant to cadmium. Furthermore, these resistant cells had 2.5-fold more metallothionein than untreated control cells. This suggests that cadmium could act to select for apoptotic-defective cells in vivo, thereby increasing the likelihood of tumor formation. This work represents the first description of cadmium affecting oncogene expression in a human cell model of a potential in vivo target site of cadmium carcinogenesis.

Apoptosis↗

PubMed2000

Possible role of caspase-3 inhibition in cadmium-induced blockage of apoptosis.

Cadmium (Cd) and chromium (Cr) are human carcinogens. Cr(VI) is taken up into cells and reduced by cellular reductants to the potential DNA damaging species Cr(V), (IV), and (III). Reactive oxygen species and carbon-based radicals may also be produced during Cr reduction. We previously found that Cd blocks Cr-induced apoptosis, which could allow a larger proportion of genetically damaged cells to escape and become transformed. This study helped define the mechanisms of Cd-induced suppression of apoptosis. Chinese hamster ovary (CHO K1-BH4) cells were treated with either Cd (5-20 microM), Cr(VI) (350 microM), or Cd (5-20 microM) plus Cr(VI) (350 microM) for 3 h and then cultured in metal-free media for an additional 48 h at which time DNA was extracted or nuclei were examined to determine apoptosis. Cd markedly reduced Cr-induced DNA fragmentation and reduced the number of Cr-induced apoptotic cell nuclei to control levels. Additional study investigated the biokinetics and cellular metabolism of Cr. Cd did not alter the cellular Cr accumulation and there were no differences in the levels of reduced glutathione, a compound possibly important in Cr reduction and reflective of the cellular reducing environment. The antiapoptotic effect of Cd was not due to diminished cellular reduction of Cr(VI) as assessed by electron-spin resonance determination of the levels of Cr(V). Thus, Cd suppression of Cr-induced apoptosis is not based on altered Cr toxicokinetics or metabolism. In addition to Cr, Cd also inhibited apoptosis induced by hygromycin B and actinomycin D. Cd was a very effective inhibitor of caspase-3 activity, a central mediator of apoptosis, with nontoxic levels of Cd resulting in up to approximately 60% inhibition. These results indicate that Cd may have a generalized inhibitory effect on apoptosis, possibly by inhibiting caspase-3. Inhibition of apoptosis by Cd may allow a greater portion of genetically damaged cells to survive, or give selective growth advantages, and has implications as a potential nongenotoxic mechanism of Cd carcinogenesis.

Animals↗

PubMed2000

Lead effects on protamine-DNA binding.

BACKGROUND: Lead impairs male fertility and may affect offspring of exposed males, but the mechanisms for this impairment are not completely clear. Protamine P1 and P2 families pack and protect mammalian sperm DNA. Human HP2 is a zinc-protein and may have an important role in fertility. As lead has affinity for zinc-containing proteins, we evaluated its ability in vitro to bind to HP2 and its effects on HP2-DNA binding. Methods and Results UV/VIS spectroscopic data indicated that HP2 binds both Pb(2+) and Zn(2+)(as chloride salts). They also provided evidence that thiol groups mainly participate for Zn(2+)-binding; however, HP2 has additional binding sites for Pb(2+). The mobility shift assay showed that lead interaction with HP2 caused a dose-dependent decrease on HP2 binding to DNA, suggesting that lead may alter chromatin stability. CONCLUSIONS: These in vitro results demonstrate that lead can interact with HP2 altering the DNA-protamine binding. This chemical interaction of lead with protamines may result in chromatin alterations, which in turn may lead to male fertility problems and eventually to DNA damage.

Chromatin↗