p,p'-DDT induces microcytic anemia in rats.

Emerging evidence suggests that chronic exposure to DDT and its derivatives is associated with a variety of human disorders such as anemia. The present study demonstrated that p,p'-DDT caused microcystic anemia in a dose-dependent manner (0, 5, 50, and 500 ppm) in the long-term study up to 2 years. To elucidate the mechanism(s) by which p,p'-DDT induces anemia, certain hematological parameters were assessed in rats fed specific doses of p,p'-DDT for 2 weeks, and the effect of lipopolysaccharide on anemia of inflammation was also examined in p,p'-DDT-treated rats. The parameters included the content of hemoglobin per reticulocyte, mean corpuscular volume of reticulocytes and mature erythrocytes, corpuscular hemoglobin concentration mean of mature erythrocytes, and saturation levels of transferrin and iron. During the 2-week treatment period, hypochromic microcytic reticulocytes and hypochromic normocytic mature erythrocytes were observed in p,p'-DDT-treated rats, with no evidence of alteration in plasma iron levels. p,p'-DDT enhanced microcytosis of reticulocytes, as well as mature erythrocytes, which occurred due to severe hypoferremia resulting from anemia of inflammation; however, plasma iron levels were attenuated probably through the inhibition of interleukin-6. Our data suggests that long-term treatment with p,p'-DDT induces microcytic anemia, possibly because of the impairment of iron utility in erythrocytes.

Malignant peritoneal mesothelioma with a sarcomatoid growth pattern and signet-ring-like structure in a female f344 rat.

We report a biphasic malignant mesothelioma in an aged female F344/DuCrlCrlj rat. Macroscopically, multiple pale brown nodules were observed in the abdominal cavity with retention of bloody ascites. Histopathologically, the tumor cells spread over the peritoneum and formed masses on the surface and underlying adipose tissues. The tumor cells dominantly proliferated in a solid, nodular or nest-like pattern with modest amount of fibrillar connective tissues, which contained hyaluronan. The tumor consisted of ovoid, polygonal or spindle-shaped cells that possessed eosinophilic cytoplasms including glycogen; some tumor cells showed a signet-ring-like structure. Multinucleated cells and mitosis were found frequently, and direct invasion to intra-abdominal organs and intravascular metastasis to the liver were observed. Immunohistochemically, keratin and mesothelin were strongly positive in most of tumor cells, while vimentin was mainly positive in spindle-shaped cells. Podoplanin was also positive, particularly in the cell membrane of tumor cells. Electron microscopically, tumor cells showed an intercellular desmosome-like structure, basement membrane and microvillus. We diagnosed the case as a malignant peritoneal mesothelioma with a sarcomatoid growth pattern and signet-ring-like structure.

Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF-ß signaling in mice.

Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor-ß (TGF-ß) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF-ß signaling. In order to further evaluate TGF-ß signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased α-smooth muscle actin-positive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-ß1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-ß1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 - a TGF-ßRI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF-ß signaling.

Immunotoxicity of the organochlorine pesticide methoxychlor in female ICR, BALB/c, and C3H/He mice.

Several types of pesticides, including organochlorines, are known to suppress or modulate immune responses. The present study evaluated the immunotoxicity of the organochlorine pesticide methoxychlor (MXC) in female BALB/c, C3H/He, and ICR mice. Mice were given oral MXC doses of 0, 30, 100, and 300 mg/kg each day for 7 consecutive days. On day 4, the mice also received an intravenous injection of sheep red blood cells (SRBC). The splenic plaque-forming cell (PFC) IgM response and the serum anti-SRBC IgM antibody titer were evaluated while splenic lymphocytes were counted by flow cytometry and the spleen underwent histopathological analysis. Significant decreases in IgM PFC responses were seen in BALB/c, C3H/He, and ICR mice that received MXC doses of 100 and 300 mg/kg. Similar changes in serum anti-SRBC IgM antibody titers occurred in three strain mice. Flow cytometric analysis revealed significantly decreased splenic T-cell (CD3+) populations in a dose dependent manner in BALB/c mice, and in the 300 mg/kg of MXC-treated group of C3H/He mice. Germinal center (GC) B-cell (CD19+PNA+) populations were significantly decreased in the 300 mg/kg of MXC-treated groups of all three mouse strains and in the 30 and 100 mg/kg of MXC-treated groups of BALB/c and C3H/He strain mice. Histopathological analysis revealed decreased cellularity of the periarteriolar lymphoid sheath (PALS; T-cell area) and decreased GC development in all three strains of mice treated with 300 mg/kg MXC. These results suggest that MXC has an immune-suppressive effect in mice, and that our protocol may be useful for rapidly detecting immunosuppression induced by environmental chemicals.

Two-generation reproduction toxicity study in rats with methoxychlor.

A two-generation reproduction toxicity study was conducted in rats with a reference estrogenic pesticide, methoxychlor, to validate the sensitivity and competency of current guidelines recommended by the United States Environmental Protection Agency; Japanese Ministry of Agriculture, Forestry and Fisheries; and Organisation for Economic Co-operation and Development for predicting reproductive toxicity of the test compound based on estrogenic endocrine disrupting effects. Both sexes of SD rats were exposed to methoxychlor in the diet at concentrations of 0, 10, 500 and 1500 ppm for two successive generations. The present study has successfully detected estrogenic activities and reproductive toxicities of methoxychlor, as well as its systemic toxicity. Body weights, body weight gains and food consumption of both sexes of animals were suppressed significantly in the 500 and 1500 ppm groups. Typical reproductive toxicities observed in females of these groups included, but were not limited to, prolonged estrous cycle, reduced fertility, decreased numbers of implantation sites and newborns, decreased ovary weights and/or increased incidences of cystic ovary. Uterine weights of weanlings increased significantly in these groups, suggesting that the sensitivity of this parameter for predicting estrogenic ability of the test compound is comparable to that of the uterotrophic assay. Reproductive toxicities of methoxychlor seemed less potent in males than in females. Methoxychlor delayed preputial separation and significantly reduced sperm counts and reproductive organ weights of males of the 500 and/or 1500 ppm groups; however, most males that failed to impregnate females in the same group showed normal fertility when they were re-mated with untreated females. Neither systemic nor reproductive toxicities appeared in the 10 ppm group.

The enhancing effect of the antioxidant N-acetylcysteine on urinary bladder injury induced by dimethylarsinic acid.

Dimethylarsinic acid (DMA(V)), the major excreted metabolite of inorganic arsenic, is carcinogenic to the rat urinary bladder. Oxidative stress has been proposed as one possible mechanism of DMA(V)-induced carcinogenesis. The authors determined whether the antioxidant N-acetylcysteine (NAC) modifies DMA(V)-induced urinary bladder injury in rats. The treatment solutions--DMA(V) at 10 mg/kg, NAC at 90 or 1.6 mg/kg (high or low dose, respectively), and their combination--were intravesically instilled into female F344 rats over two hours under pentobarbital anesthesia. The treatment was conducted twice with an interval of three days. All animals were euthanized one day after the second treatment. NAC (low dose) alone did not induce histopathological changes or increase 5-bromo-2'-deoxyuridine (BrdU) labeling index in urothelial cells. Both DMA(V) and NAC (high dose) induced a weak neutrophil infiltration and an increase in the BrdU labeling index; these pathological changes were enhanced by the combined treatment of DMA(V) and NAC (high or low dose). Increased oxidative stress and urothelial cell hyperplasia with evidence of activated p44/42 MAPK (ERK1/2) and cyclin D1 were found in the DMA(V) and NAC (high dose) cotreated group. These results suggest that cotreatment with NAC enhanced DMA(V)-induced urinary bladder injury and that the effects may be mediated by excess oxidative stress and ERK signaling.

Altered pulmonary defense system in lung injury induced by didecyldimethylammonium chloride in mice.

Didecyldimethylammonium chloride (DDAC), a representative dialkyl-quaternary ammonium compound (QAC), could contaminate working atmospheres when used in disinfectant operation and adversely affect human health. Furthermore, the development of bacteria resistant to DDAC might become public health concern. We postulated that DDAC instillation in the lungs alters pulmonary antioxidant and antimicrobial responses and increases susceptibility to systemic administration of a bacterial component lipopolysaccharide (LPS). Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment. Pulmonary cytotoxicity in recovered bronchoalveolar lavage was evident on Days 1 and 7, and inflammatory cell influx and interleukin-6 expression peaked on Day 7, in association with altered antioxidant and antimicrobial responses, as demonstrated by measuring heme oxygenase-1, glutathione peroxidase 2, lactoferrin, and mouse ß-defensin-2 and -3 mRNA in the lung samples. The impaired defense system tended to enhance the inflammatory reaction caused by a systemic administration of LPS; the effect was in association with increased expression of toll-like receptor-4 mRNA. The results suggest that DDAC alters pulmonary defense system, which may contribute to susceptibility to an exogenous infectious agent.

Malignant Leydig cell tumor with spindle-shaped cells in a male CD-1 mouse.

Leydig cell tumors with spindle-shaped cells are very rare in humans and animals. We report that an 84-week-old male CD-1 mouse had a malignant Leydig cell tumor characterized by proliferation of oval to spindle-shaped cells with or without fat deposition, and with a storiform pattern. These cells were immunopositive for inhibin and S-100, and negative for the androgen receptor, thereby suggesting that they may have differentiated from Leydig cells. This differentiation from Leydig cells was further confirmed by the immunopositivity of these cells for nestin and alpha-smooth muscle actin, both of which are known to be expressed in the stem/progenitor cells that differentiate into Leydig cells. These findings suggest that the tumor is most probably a malignant spindle-cell-type Leydig cell tumor.

Pulmonary injury and antioxidant response in mice exposed to arsenate and hexavalent chromium and their combination.

Chromated copper arsenate, which is used worldwide as a wood preservative, can adversely affect human health. Accumulating evidence suggests that chromium (Cr) and arsenic (As) can potentially disrupt the redox balance and cause respiratory diseases and cancer in humans. The present study was designed to determine the combined toxic effects of these metals in the lungs and to clarify the specific molecules that are stimulated by combined exposure to both metals. Male C57BL/6J mice were intratracheally instilled with arsenate [As(V)], hexavalent chromium [Cr(VI)], or a combination of both metals. Mice were sacrificed 2 days after treatment to collect bronchoalveolar lavage fluid and lung tissue samples. Inflammation, cytotoxicity, apoptosis, and oxidative stress markers were measured. Our results indicated that administration of Cr(VI) alone or in combination with As(V) induced neutrophil-dominant inflammation as well as phosphorylation of mitogen-activated protein kinases; effects of treatment with As(V) alone were comparatively less potent. By analyzing the production of interleukin-6 and activity of lactate dehydrogenase and caspase, we confirmed that co-treatment intensified pulmonary injury and that it was accompanied by oxidative stress, as confirmed by marked increases in the production of reactive oxygen species, reduced glutathione content, and thioredoxin reductase (TRXRD) activity. Expressed mRNA levels of heme oxygenase-1, glutamylcysteine ligase, glutathione peroxidase 2, thioredoxin (TRX) 1, and TRXRD1 were also enhanced by co-treatment, whereas treatment with As(V) alone reduced the mRNA expression level of TRX2. Our data suggest that co-treatment with As(V) exacerbated Cr(VI)-induced pulmonary injury and that this effect may be exerted through a disruption in the balance among several antioxidant genes.

The effect of testosterone propionate supplement on testicular toxicity with thiamphenicol in male Sprague-Dawley rats.

Testosterone propionate (TP) was supplemented to male rats for assessment of its ameliorating effect on testicular toxicity with thiamphenicol (TAP). A total of 20 male Sprague-Dawley rats were treated orally with TAP at 200 mg/kg/day for up to 4 weeks. In addition, 5 male rats were allotted to the control group receiving vehicle only. Ten of the 20 treated rats had a Silastic capsule (containing about 80 mg of TP) implanted in the dorsal skin at Week 2 and assigned to the TAP-TP group, while the other 10 treated rats were in the TAP group. After Weeks 3 and 4, five of both treated groups were examined for weight and histology of the testis and accessory genital glands, and for staging analysis of the seminiferous tubules. The same parameters were also assessed in the control group after Week 4. Weights and morphology of the seminal vesicle and prostate recovered remarkably from the TAP toxicity after TP supplement. However, no ameliorating effects of TP were obtained for the testis in either weight, morphology, or staging analysis of the seminiferous tubules.