M. Ban

Role of γ-glutamyltranspeptidase and β-lyase in the nephrotoxicity of hexachloro-1,3-butadiene and methyl mercury in mice

Abstract Male Swiss OF1 mice received a single oral dose of either 80 mg kg hexachloro-1,3-butadiene (HCBD) or 80 mg kg methyl mercury (MeHg). Examination of cryostat kidney sections stained for alkaline phosphatase (APP) revealed damage to about 50% of the proximal tubules after 8 h. Pretreatment with the γ-glutamyltranspeptidase (γ-GT) inactivator AT-125 (Acivin, 50 mg kg i.p., plus 50 mg kg p.o., reduced the number of damaged tubules by 59 and 58% in mice treated with HCBD and MeHg, respectively. Pretreatment with the two β-lyase inhibitors, amino-oxyacetic acid (AOAA, 3 × 100 mg kg p.o.) and dl -propargylglycine (PPG, 300 mg kg i.p. plus 300 mg kg p.o), reduced HCBD nephrotoxicity by 46 and 59%, respectively, but did not protect against MeHg nephrotoxicity. The results support a role for γ-GT and β-lyase in the mouse renal toxicity of HCBD and implicate γ-GT but not β-lyase in MeHg-induced nephrotoxicity in mice.

Iron oxide particles modulate the ovalbumin-induced Th2 immune response in mice

This study was designed to investigate the modulatory effects of submicron and nanosized iron oxide (Fe(2)O(3)) particles on the ovalbumin (OVA)-induced immune Th2 response in BALB/c mice. Particles were intratracheally administered four times to mice before and during the OVA sensitization period. For each particle type, three different doses, namely 4×100, 4×250 or 4×500 μg/mouse, were used and for each dose, four groups of mice, i.e. group saline solution (1), OVA (2), particles (3), and OVA plus particles (4), were constituted. Mice exposed to OVA alone exhibited an allergic Th2-dominated response with a consistent increase in inflammatory scores, eosinophil numbers, specific IgE levels and IL-4 production. When the mice were exposed to OVA and to high and intermediate doses of iron oxide submicron- or nanoparticles, the OVA-induced allergic response was significantly inhibited, as evidenced by the decrease in eosinophil cell influx and specific IgE levels. However, the low dose (4×100 μg) of submicron particles had no significant effect on the OVA allergic response while the same dose of nanoparticles had an adjuvant effect on the Th2 response to OVA. In conclusion, these data demonstrate that the pulmonary immune response to OVA is a sensitive target for intratracheally instilled particles. Depending on the particle dose and size, the allergic response was suppressed or enhanced.

Relationship between IgE positive cell numbers and serum total IgE levels in mice treated with trimellitic anhydride and dinitrochlorobenzene.

Although increased total serum IgE levels have been suggested as being predictive parameters of airway hypersensitivity caused by low molecular weight chemicals, it is not yet clear what level of serum total IgE in chemically-treated animals would translate to potential risk of inducing an immediate type hypersensitivity in human beings. Quantitative determination of IgE-bearing positive cells induced by chemicals in the tissue, particularly in respiratory airway, could help to resolve this problem. In BALB/c mice, serum total IgE concentrations and tissue IgE-bearing positive cell numbers were investigated following topical exposure to the chemicals, trimellitic anhydride (TMA) and dinitrochlorobenzene (DNCB), known in human as a respiratory and dermal sensitizer, respectively. In groups of mice 8 and 10 weeks of age, there were less individual variations in 25% TMA-induced serum total IgE increase than in other groups of mice 6, 12 and 16 weeks of age. When chemical concentrations of 1, 2 and 3% DNCB and of 6.25, 12.5 and 25% TMA were studied, we observed that the increase in IgE was dose-dependent for both chemicals, r=0.96; P=0.03 for DNCB and r=0.99; P=0.002 for TMA. However, the increase in serum total IgE induced by TMA was at least twice that induced by DNCB and was associated with the numbers of IgE bearing cells in the tracheal lamina propria (r=0.67, P=0.0003). A respiratory hypersensitivity caused by TMA, but not by DNCB, may be partly due to the presence of immuno effector cells bearing IgE at their surface in airway microenvironments, and their presence might be related to the higher level of serum total IgE. The IgE bearing positive cells could, therefore, help to identify chemicals which have the potential risk of inducing an immediate type hypersensitivity in humans.

EFFECT OF SUBMICRON AND NANO-IRON OXIDE PARTICLES ON PULMONARY IMMUNITY IN MICE

Due to advances in nanotechnology, exposure to particle compounds in the workplace has become unavoidable. Assessment of their toxicity on health is an important occupational safety issue. This study was conducted in mice to investigate the toxicological effects of submicron and nano-iron oxide particles on pulmonary immune defences. In that purpose, we explored for the first time, inflammatory and immune responses in lung-associated lymph nodes. For each particle type, mice received either a single intratracheal instillation at different concentrations (250, 375, or 500μg/mouse) or four repeated instillations at 500μg/mouse each. Cytokine production, inflammatory and innate immune response, and humoral immune response were respectively assessed 1, 2, and 6 days after particle exposures. Both types of particles induced lung inflammation associated with increased cytokine productions in lymph node cell cultures and decreased pulmonary immune responses against sheep erythrocytes. Natural killer activity was not modified by particles. In comparison to single instillation, repeated instillations resulted in a reduction of inflammatory cell numbers in both bronchoalveolar lavages and pulmonary parenchyma. Moreover, the single instillation model demonstrated that, for a same dose, nano-iron oxide particles produced higher levels of inflammation and immunodepression than their submicron-sized counterparts.

Role of tachykinins and neutral endopeptidase in toluene diisocyanate-induced bronchial hyperresponsiveness in guinea pigs

The role of tachykinins in toluene diisocyanate (TDI)-induced non-specific bronchial hyperreactivity (NSBH) in guinea pigs was investigated, and it was determined whether or not the activity of airway neutral endopeptidase (NEP) was inhibited in conditions where a bronchial hyperreactivity to acetylcholine (ACh) was observed. Exposures to 3 ppm TDI for 1 h, or to 0.029 ppm for 8 weeks caused a significant bronchial hyperreactivity to ACh. The depletion of tachykinins by a pretreatment with capsaicin (140 mg/kg) eliminated the TDI-induced airway hyperresponsiveness in both patterns of exposure to TDI. Capsaicin treatment had no effect on the response to ACh in guinea-pigs exposed to air (controls). Bronchial NEP activity determined by histoenzymology was significantly less 4 and 24 h after the end of a 1-h exposure to 3 ppm TDI than after exposure to air. Bronchial NEP activity evaluated 24 h after the end of a 48-h exposure to 0.116 ppm TDI, or a 1-week exposure to 0.050 ppm TDI was not significantly different from those of controls exposed to air, whereas in the same conditions of exposure a NSBH is observed in guinea-pigs. These data suggest that tachykinins released from C-fibers upon acute or repeated exposures to high or low concentrations of TDI, respectively, play an essential role in the observed bronchial hyperreactivity, and that the inhibition of NEP by TDI cannot completely account for the observed airway hyperreactivity.

Acetone compared to other ketones in modifying the hepatotoxicity of inhaled 1,2-dichlorobenzene in rats and mice.

The ability of acetone and 3 other ketone vapours to influence the hepatotoxicity of inhaled 1,2-dichlorobenzene (DCB) was examined in rats and mice. Methylethylketone, methylisobutylketone or cyclohexanone increased liver cytochrome P-450 content and glutathione-S-transferase (GST) activity, but did not affect serum glutamate dehydrogenase (GLDH) activity in rats. Pre-exposure to these ketones enhanced DCB-induced increase in serum GLDH activity (8-63-fold), while the increases in cytochrome P-450 content (33-86%) and GST activity (42-64%) were identical to those resulting from exposure to ketones alone. Each of the 3 levels of exposure to acetone elicited cytochrome P-450 and GST responses comparable with those caused by the other ketones. In spite of that, acetone pre-exposure potentiated (4785 ppm), reduced (10670 ppm) or suppressed (14790 ppm) DCB-induced liver toxicity. In mice, the 3 ketones mentioned above interacted with DCB on centrolobular liver glucose-6-phosphatase (G-6-Pase) while acetone pre-exposure elicited an interactive G-6-Pase response in the mediolobular area alone, suggesting topographic change.

Probenecid-induced protection against acute hexachloro-1, 3-butadiene and methyl mercury toxicity to the mouse kidney

Male Swiss OF1 mice received a single oral dose of either 80 mg/kg hexachloro-1,3-butadiene (HCBD) or 40 mg/kg methyl mercury (MeHg). Examination of cryostat kidney sections stained for alkaline phosphatase (APP) revealed damage to about 50% of the proximal tubules after 8 h. Treatment with the organic anion transport inhibitor probenecid (i.p., 3 x 0.75 mmol/kg) did not have any renal effect in normal mice but reduced the number of damaged tubules by 80 and 90% in mice treated with HCBD and MeHg respectively. The results support the conclusion that the toxicity of HCBD and MeHg to the mouse kidney is related to a probenecid-sensitive transport process. It cannot be stated from the present investigation whether the inhibition nephrotoxicity data are related to classic organic anion secretion by the kidney.

Effects of nickel sulfate on pulmonary natural immunity in Wistar rats

This study was designed to investigate the in vivo effect of nickel sulfate on the pulmonary non-specific immune defences. Groups of four male Wistar rats were treated with a single intratracheal instillation of NiSO(4) at different doses: 1, 2, 4 and 8 micromol of NiSO(4) per rat. Control rats received a corresponding instillation of the saline vehicle. The effect of NiSO(4) on the cytotoxic activity of the pulmonary natural killer (NK) cells and alveolar macrophages (AM), as well as the pulmonary production of cytokines such as alpha-tumor necrosis factor (TNF-alpha) and gamma-interferon (IFN-gamma), were examined 1, 2 and 7 days later. Spontaneous NK-cytotoxicity towards mouse-derived tumor cell line, Yac-1 was suppressed 1 day after treatment at doses of 2 micromol/rat and above with only one result significant (P<0.05); 2 days after treatment the suppression was increased with all results significant at the same doses; 1 week after treatment NK activity restoration was observed except for the highest dose, 8 micromol/rat. AM-mediated cytotoxicity towards mouse-derived tumor cell line, 3T12, did not show any significant difference in treated and untreated animals. In contrast, whereas moderate levels of TNF-alpha were detected in the broncho-alveolar lavage (BAL) fluid supernatants of controls, the NiSO(4) treatment highly suppressed TNF-alpha production with a maximum observed after 2 days. TNF-alpha suppression was found to be transient, at least with the lowest NiSO(4) dose, with levels returning to normal after 7 days. A non-significant increase in IFN-gamma was observed in the BAL fluids of treated animals at each time of examination. Taken together, these results indicate that NK cell activity and TNF-alpha secretion are sensitive targets for instilled NiSO(4) in Wistar rats.

TDI inhalation in guinea-pigs involves migration of dendritic cells.

Toluene diisocyanate (TDI) can cause occupational asthma, but the mechanism underlying sensitization to this chemical compound remains controversial. The present study aims to investigate whether tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) liberated in the lungs after TDI inhalation can contribute to the migration of dendritic cells from respiratory airways towards lung associated lymph nodes for presentation of TDI hapten. Exposure was studied in two modes: (1) acute exposure (experiment no. 1, 2 and 3) where animals were exposed to 2.962, 1.060, and 1.076 ppm TDI for 1, 4, and two periods of 4 h, respectively; (2) subacute exposure (experiment no. 4, 5 and 6) where animals were exposed to 0.066, 0.110, and 0.999 ppm TDI for 48 h for the two lower doses and 5 days for the highest dose. Depending on the modes of exposure, two to four post exposure times were selected. After acute exposure to 2.962 ppm TDI for 1 h, the increase in TNF-alpha and IL-6 levels in bronchoalveolar lavage (BAL) fluid was observed immediately at the end of inhalation exposure, whereas the maximum number of dendritic cells and total cells occurred at post exposure times of 48 h and 5 days, respectively. In two other acute exposures, the peak increases in TNF-alpha, IL-6 and total cell numbers were observed at 48 h post exposure time, whereas the peak increase in dendritic cells occurred at 24 h. After subacute exposure to 48 h TDI, where TDI concentrations were relatively low (0.006 or 0.110 ppm), a parallel increase in TNF-alpha and IL-6 levels, dendritic and total cell numbers were observed at 0 h post exposure time. This phenomenon was also apparent at 24 h post exposure time when the animals had been exposed to 1.999 ppm TDI for 5 days. From these results, we can conclude that dendritic cells could play a key role as antigen presenting cells in the development of TDI-induced respiratory sensitization, and that their migration toward lung-associated lymph nodes is probably conditioned by cytokine release in their micro-environment. Future work must delineate whether TNF-alpha and IL-6 are solely responsible for the migration of dendritic cells after TDI inhalation, for example by using antibodies to neutralize these cytokines.

Induced hepatotoxicity in female rats by aflatoxin B1 and ethynylestradiol interaction

Female Sprague-Dawley rats were treated with a single ip dose of aflatoxin B1 (AFB1) (3 or 6 mg/kg). Twenty-four hours later and weekly until killed, some of the rats treated with AFB1 were given ethynylestradiol (EE) by gavage at the dose of 13 mg/kg. One, three, six, and nine months following the beginning of the experiment, animals were killed. Light microscopy of liver and histochemical determinations of gamma-glutamyltransferase (GGT) as well as the measurement of hepatic drug-metabolizing enzyme activities were investigated. The results show that AFB1 induced only very weak changes in the levels of different constituents studied. Thus, the mycotoxin did not affect GGT activity and increased epoxide hydrolase activity by a maximum of 42%. In contrast, EE significantly and progressively decreased (20 to 50%) the activity of UDP-glucuronosyltransferase (UDPGT) as well as the concentration of cytochrome P-450 and microsomal proteins. However, the estrogen increased the activity of epoxide hydrolase up to 150% as well as the activity of the hepatic (400%) and plasma (175%) GGT. The results also indicate that AFB1 amplified the EE-induced increase in liver weight and enhanced the depressive effects of the estrogen on microsomal proteins, cytochrome P-450, and UDPGT. Foci of cellular alteration which consisted of clear, acidophilic and basophilic cell lesions were seen in the livers of treated rats examined by light microscopy. These lesions were more prominent in the livers of animals given combinations of AFB1 and EE; they were accompanied by a strong intensity of GGT staining in the periportal area and a marked increase of the enzyme activity in the plasma (324%). From the sixth month, the livers of some animals treated with the combinations of AFB1 and EE showed hyperplastic nodules. This study indicates that the interaction between chronic administration of EE and a single ip injection of AFB1 induces hepatic lesions considered as possible forerunners of liver cell carcinomas. It also shows that GGT is a potential marker of preneoplastic lesions and may be used, therefore, in epidemiologic surveys in humans exposed to liver carcinogens such as the aflatoxins.