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.

A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats.

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.

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.

Pulmonary fibrosis in response to environmental cues and molecular targets involved in its pathogenesis.

Chronic lung injury resulting from a variety of different causes is frequently associated with the develop ment of pulmonary fibrosis in humans. Although the etiology of pulmonary fibrosis is generally unknown, several sources of evidence support the hypothesis that a number of environmental and occupational agents play an etiologic role in the pathogenesis of this disease. The agents discussed in this review include beryllium, nylon flock, textile printing aerosols, polyvinyl chloride and didecyldimethylammonium chloride. The authors also describe a variety of animal models, including genetically modified mice, in order to investigate the molecular mechanism of pulmonary fibrosis, focusing on chemokine receptors, regulatory T cells and transforming growth factor-ß and bone morphogenetic protein signaling. Overall, we propose the concept of toxicological pulmonary fibrosis as a lung disease induced in response to environmental cues.

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.

Differential expression of genes encoding preprobrevinin-2, prepropalustrin-2, and preproranatuerin-2 in developing larvae and adult tissues of the mountain brown frog Rana ornativentris.

Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs. The levels of preprobrevinin-2 and preproranatuerin-2 mRNAs in the skin specimens of developing R. ornativentris larva were detectable only at stages later than the onset of metamorphosis and reached peaks at the stage of metamorphic climax. In contrast, prepropalustrin-2 mRNA was detected prior to the onset of metamorphosis and levels peaked at stages earlier than those of the other two mRNAs. In adult animals, preprobrevinin-2 and preproranatuerin-2 gene transcripts were detected at low levels in the small intestine and skeletal muscle but not in the stomach, liver, or kidney, whereas prepropalustrin-2 gene transcripts were detected at relatively high concentrations in all tissues examined. These results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation.

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.

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice.

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 µg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 µg/kg of DDAC caused weakly induced inflammation, and exposure to 15µg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 µg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1ß, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C-C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.

Developmental and thyroid hormone-induced expression of preprotemporin genes in the skin of Japanese mountain brown frog Rana ornativentris.

Temporins are a group of small, highly hydrophobic, antimicrobial peptides widely distributed in the skin of frogs from the Ranidae family. In order to examine the mechanisms of regulation of temporin gene expression, we measured expression levels of preprotemporin mRNA in the skin of the Japanese mountain brown frog Rana ornativentris, using a semiquantitative RT-PCR system. Preprotemporin mRNAs were not detected in skin prior to the onset of metamorphosis but their levels increased markedly during metamorphosis, reaching a maximum at the stages of metamorphic climax, suggesting a correlation with thyroid hormone concentrations. Consequently, we examined direct effects of triiodothyronine (T(3)) on in vivo preprotemporin gene expression. Treatment of adult animals with 2 x 10(-9) mol/L T(3) for 48 h raised the preprotemporin mRNA levels in skin by 1.5-fold compared with untreated controls.

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs.

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.

Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii.

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.

Molecular cloning and sequence analyses of preprotemporin mRNAs containing premature stop codons from extradermal tissues of Rana tagoi.

The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM).

Developmental and triiodothyronine-induced expression of genes encoding preprotemporins in the skin of Tago's brown frog Rana tagoi.

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.