The survival of parasites is dependent on that of the host. It is considered that parasites originated from nonparasitic ancestors and adapted to the environment in the host during the evolutional process, and developed host-and organ-specificities. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under a low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. In such systems, parasite mitochondria play diverse roles. Especially, marked changes in the morphology and components of the mitochondria in the life cycle are very interesting in biological aspects such as developmental control and environmental adaptation. Such unique properties of parasite mitochondria could be promising targets for chemotherapy.
The generation of superoxide anion or hydroxyl radical derived from the organic selenium compounds selenomethionine, selenoethionine, selenocystine, selenocystamine and selenocysteine-glutathione selenenyl sulfide (CySeSG) was investigated by the electron spin resonance (ESR) technique with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of ESR signals of DMPO-OOH adduct formed by the reaction of the hypoxanthine/xanthine oxidase reaction system with DMPO decreased in the presence of selenomethionine or selenoethionine. However, the decrease of this ESR signal intensity was not due to superoxide anion-scavenging ability of these selenium compounds. When selenomethionine or selenoethionine existed in the superoxide anion generating system at a higher concentration, a new ESR signal was recognized. This signal disappeared with the addition of a hydroxyl radical-scavenging reagent and was similar to the signal of DMPO-OH adduct. The lack of structural change in selenomethionine or selenoethionine following reaction with components of the superoxide anion generating system suggested that these selenium compounds act as a catalyst. Such a phenomenon was not observed in the superoxide anion generating system in the presence of selenocystine, selenocystamine or CySeSG. These findings suggested that coexistence of selenomethionine or selenoethionine under mammalian physiological conditions generating superoxide anion may possibly form a hydroxyl radical.
The suppressive effect of (-)-epigallocatechin gallate (EGCG), the major polyphenolic constituent present in green tea, on aflatoxin B1 (AFB1)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. The administration of EGCG 24 hr before the AFB1 injection significantly suppressed AFB1-induced CA. The suppression was observed 18 hr, 24 hr and 48 hr after the AFB1 treatment but no suppressive effect was observed at the early period (6 hr and 12 hr) after the AFB1 treatment. Furthermore, the suppression was observed in all doses of AFB1 (1, 5, 10 and 20 mg/kg) investigated. Rats given EGCG 2 hr before the AFB1 injection displayed no suppressive effect. The suppressive effect of EGCG paralleled the dose of EGCG when given in a dose range of 10-60 mg/kg body weight. The administration of (-)-epicatechin gallate 24 hr before the AFB1 injection significantly suppressed AFB1-induced CA as well as EGCG. On the other hand, in rats given green tea polyphenols (GTP) 2 hr before the AFB1 injection, (-)-epigallocatechin and gallic acid significantly suppressed AFB1-induced CA. The pretreatment with EGCG or gallic acid did not induce the drug-metabolizing enzymes in rat liver, such as cytochrome P450 and glutathione S-transeferase. Rats given 2% green tea infusion as the sole source of drinking water for four days before sacrifice displayed significantly suppressed AFB1-induced CA. However, rats given various kinds of canned tea for four days showed no suppressive effect. The amount of GTP in canned tea determined by high performance liquid chromatography (HPLC) analysis was much less than that in 2% green tea infusion.
To establish a useful and convenient bioassay for evaluating immunotoxicological effects of environmental chemicals, lymphocyte mitogenesis tests of about 255 chemicals and environmental water were performed. We determined the growth-inhibitory effect of the environmental chemicals on the mouse splenic lymphocyte mitogenesis using lipopolysaccharide and concanavalin A as the specific mitogen for B cells and T cells, and evaluated toxicity on humoral immunity and cell-mediated immunity, respectively. The DNA content of grown cells was determined by the novel ethidium bromide-fluorophotometry method, with 96-well microplates. Of the 255 chemicals tested, 173 chemicals showed inhibitory effects on the mitogenesis. The data were classified into four typical inhibition patterns from the dose-response curves. The chemicals were categorized into six groups in the respect of inhibition selectivity between B cell mitogenesis and T cell mitogenesis. Some chemicals, such as metallic compounds, showed nonspecific effects for B cell and T cell-mitogenesis, which was attributed to the cytotoxicity. The previous reports relating to immunotoxicity found about 123 chemicals; 78 chemicals (63.4%) were positive and one chemical (0.8%) was negative for both experimental results and reported results. In total, the concordance between experimental results and reported results is 64.2%. In the application of river water concentrates for the lymphocyte mitogenesis test, some of the samples inhibited both B cell and T cell mitogenesis at the tested volume of 7.2 ml. These results indicated that the lymphocyte mitogenesis test is applicable in estimating immunotoxicity of pollutants in environmental water as well as authentic environmental chemicals.
The number of diesel-powered vehicles has been increasing in Japan in recent years. And chronic exposure to relatively low levels of diesel exhaust may be a risk factor for respiratory diseases. It has been demonstrated that the airborne particulates sampled at a roadside contain exhaust from diesel engines. The present study has been undertaken to evaluate the cytotoxicity of airborne particulates in human and Chinese hamster lung fibroblast cell lines (WI-38 and CHL/IU). Conversion of mitochondrial MTS, leakage of lactate dehydrogenase (LDH), and the effect on cell proliferation curves were monitored. A dose–related effect of airborne particulate extracts was observed in MTS and LDH leakage assay with both cell lines. Crude extract was most cytotoxic in the MTS assay and had a significant effect on cell proliferation. The response of extracts and fractions of these extracts on MTS assay were similar to the effect on cell proliferation in both cell systems. An increase in LDH leakage was detected in all extracts and fractions except crude extract. In comparison, CHL/IU proved to be more sensitive than WI-38 in MTS assay, but most extracts and fractions were more toxic to WI-38 in LDH leakage assay. These results suggest that even a small amount of substances in airborne particulates was toxic to both cell systems.
Many environmental chemicals (xenobiotics), including polychlorinated ethylenes (CEs), are known to be metabolically intoxicated via the formation of hazardous intermediates (referred to as biological activation), while the expression of metabolic enzymes might be interfered with by xenobiotics at various stages in a system to minimize their adverse biological effects on the host animals. Three CEs, i.e., tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (1, 1-DCE), were comparatively studied for their effects on the in vivo expression of CYP forms, which are responsible for their metabolic activation, as well as organospecificities between the liver and lung. Furthermore, their effects on the expression of CYP forms were studied in the animals simultaneously administered with phenobarbital (PB), known as an inducer of CYP2B. Individual CEs were administered intraperitoneally at 0.5 g/kg alone or simultaneously with PB (80 mg/kg/d) to 7-week-old male Wistar rats weighing about 200 g. The testosterone hydroxylase activities, CYP2B- and 2E1-mRNA, and CYP2B- and 2E1-proteins were measured 24 hr after the treatment. Testosterone 2β-hydroxylase (2βTSH) and 16βTSH activities are attributable to the functions of CYP3A2, and CYP2B plus CYP3A2, respectively. The induction of hepatic CYP2B in the PB-treated animals might be masked by the suppressive effect of CYP3A2 in terms of the 16βTSH activity, while the pulmonary 16βTSH activity was confined to the function of CYP2B, which was insensitive to the inducing effect of PB. The inhibition of hepatic 16βTSH activity was observed exclusively in the presence of 1, 1-DCE, especially in PB-coadministered animals, suggesting the preferable suppression of CYP2B. In the lung, however, PCE suppressed the 16βTSH activity in the absence of PB. The expression levels of hepatic CYP2B mRNA and protein were significantly lowered by 1, 1-DCE in the presence of PB. Concerning hepatic CYP2E1, no alternation was observed in the levels of mRNA and protein by any of the CEs in the absence or presence of PB, except for a marked decrease in the amount of mRNA when the rats were treated with 1, 1-DCE in combination with PB. In the lung, both mRNA and protein were not detected under the given assay conditions, as in our previous studies. Based on these results, it is suggested that 1, 1-DCE suppresses the induction of hepatic CYP2B and 2E1 in advance of the transcriptional stage. The expression of pulmonary CYP2B was obstructed by PCE posttranslationally in the absence of PB.
Human helper T cells are divided into T helper (Th) 1 and Th2 cells, which are known to have important roles in cell-mediated and humoral immunity, respectively. In the present study, we attempted to identify cell markers that can distinguish Th1 and Th2 cells using large-scale two-dimensional electrophoresis. Several proteins were found to be specifically and reproducibly expressed in each helper T cell subtype. Three proteins, which have molecular weights of 35000 (spot 1), 40000 (spot 2) and 49000 (spot 3) with pIs of 7.0, 7.0 and 6.3, respectively, were found only in Th1 cells, and the other three proteins, which have molecular weights of 32500 (spot 4), 38000 (spot 5) and 44000 (spot 6) with pIs of 6.5, 6.0 and 5.8, respectively, only in Th2 cells. Spots 1, 3 and 4 were found in both the membrane and the cytosol. Spots 2 and 5 were membrane proteins, while spot 6 was a cytosol protein. The molecular weight of spot 4 resembled that of ST2, which was reported to be a specific cell surface marker of Th2 cells. However, anti-ST2 antibody did not react with spot 4 at all. These results suggest that the proteins found in the present study are novel cell markers of Th1 and Th2 cells.
Advanced glycation end products (AGEs), generated by the Maillard reaction, accumulate in long-lived proteins like collagen and are presumed to be involved in aging and the pathogenesis of complications in diabetes. We investigated the effect of peptide-free forms of AGEs that can be released by the catabolism of AGE-proteins on cellular activities using cultured cells and synthetic AGEs, pentosidine and pyrraline. The exposure of cultured fibroblasts to 15 or 30 μM of pentosidine resulted in the partial inhibition of cell proliferation and production of extracellular matrix (ECM) proteins. Pentosidine also inhibited the proliferation and ECM protein production of chondrocytes, but its effect on cell proliferation was weak while that on ECM production was prominent, as compared with the effect of pentosidine. A similar inhibitory effect on cell proliferation was observed when fibroblasts or chondrocytes were exposed to pyrraline. Pentosidine and pyrraline did not cause cell death in these cells. Pentosidine appeared to enter the cells during the culture. These results suggest that peptide-free forms of AGEs affect cell growth and cell metabolism by acting both extracellularly and intracellularly.
Male Wistar rats were exposed to 0.2 ppm ozone for up to 14 days, during which alveolar macrophages were collected by pulmonary lavage to assess the effect of ozone on their microbial killing and superoxide-producing activities. For rapid assessment of microbial killing activity, we measured the release of 3H-radioactivity into the supernatant by deoxycholate-lysis of the macrophages that had phagosytosed and killed 3H-uridine-labeled microbes. The killing activity against Escherichia coli and Candida albicans was reduced to 70-80% of control levels on day 3. However, phagocytosis by and the activity of lysosomal enzymes of the macrophages were not impaired. On day 14 the killing activity against E. coli had returned to control levels, whereas that against C. albicans was still reduced. Because active oxygen species plays an important role in microbial killing activity of macrophages, the effects of ozone on respiratory burst and superoxide production were examined. Aliquots of alveolar macrophages were stimulated with phorbol myristate acetate (PMA), opsonized zymosan, or lipopolysaccharide (LPS) plus cytochalasin E (Cyt.E). The respiratory burst, oxygen consumption for rapid superoxide production, was decreased to 60-80% of control levels on day 3. On day 14, the respiratory burst by opsonized zymosan was still 80% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In addition, the superoxide-producing activity of ozone-exposed macrophages was 10-60% decreased on day 3. On day 14, the superoxide production by stimulation with opsonized zymosan was still 60% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In conclusion, because of their decreased production of superoxide, the host defense activity of alveolar macrophages was impaired by in vivo exposure to 0.2 ppm ozone. In particular, the C. albicans-associated defect lasted throughout the exposure period.
Metallothionein (MT), which is a low-molecular weight, cysteine-rich, metal-binding protein, is induced during acute-phase reactions. However, the specific function of MT in the acute-phase response remains to be elucidated. We previously reported that MT-I, II deficient (MT-null) mice are highly sensitive to the lethal effects of lipopolysaccharide (LPS)/D-galactosamine (GalN). We designed the present study to clarify the major cause of the differences in the sensitivity to the lethal effects of LPS/GalN between wild type and MT-null mice. We found that histological grade of hepatocellular necrosis, induced by LPS/GalN, was greater in MT-null mice than in wild type mice. Therefore, the present findings suggest that MT induction has the potential as an attenuator of LPS/GalN-induced liver necrosis
The effect of ISP-I/myriocin on adenosine 3'-monophosphate (3'-AMP) forming enzyme activity in carbon tetrachloride (CCl4)-induced hepatitis was examined. The 3'-AMP forming enzyme activity in mouse liver was enhanced by the treatment of CCl4. The increase in the activity in cytosol may result from the leakage of the enzyme from mitochondria via the damage of mitochondrial membrane induced by CCl4. Pretreatment of mice with ISP-I/myriocin for 24 hr, significantly inhibited both the CCl4-induced activities of serum alanine aminotransferase and the 3'-AMP forming enzyme in the supernatant of liver homogenate prepared immediately after the organ excision. These results suggest that the inhibitory effects of ISP-I/myriocin may be due to inhibition of lipid peroxidation of intracellular membranes caused by CCl4, in turn inhibiting the leakage of the 3'-AMP forming enzyme from mitochondria to cytosol. Our results demonstrated for the first time that ISP-I/myriocin has a protective effect on the CCl4-induced hepatotoxicity.
The migration dialkyl phthalate was tested in volunteers who chewed polyvinyl chloride (PVC) toy products under controlled conditions. The PVC toy samples consisted of ball A containing 100 and 185 mg/g di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) respectively, and ball B, containing 256 mg/g diisononyl phthalate (DINP). The migration of dialkyl phthalate into simulated saliva was also tested in vitro by shaking toy samples. The migration rates of DBP, DEHP and DINP from balls A and B were 11.7, 44.4 and 78.0 μg/10 cm2/hr, respectively, in vivo, and 339, 315 and 535 μg/10 cm2/hr, respectively, in vitro. The presence of mono-n-butyl phthalate (MBP) and mono-2-ethylhexyl phthalate (MEHP) in saliva collected after chewing ball A was confirmed by GCMSSIM. Human saliva which collected from volunteers incubated with DBP and DEHP at 37°C over 60 min, hydrolyzed these compounds to their monoesters.
Substantial evidence indicates that mental stress has adverse effects on serum lipid levels and cardiovascular health. This study examined the effects of final medical degree examinations (mental stress) on serum lipid and lipoprotein cardiovascular risk factors in African medical students. Twenty-seven healthy male and female medical students had lipids, lipoproteins, urea and uric acid assessments before and during final year examination. The results showed that black African medical students had adverse lipoprotein changes characterized by reduction in the levels of high density lipoprotein-cholesterol (HDLC) and HDLC/total cholesterol (TC) ratio [coronary heart disease (CHD) risk predictor index] during examinations, while mean urea level decreased from the baseline. These findings provide an opportunity for better planning of the nutritional requirements (vis-à-vis increase in protein intake) for medical students during examinations. They will also guide academic curriculum planners on how to work out strategies to reduce mental stress and its associated metabolic disturbances among the students during examinatinos, throughout the period of their medical training. In all, these findings will add to our knowledge of the numerous factors that may affect lipid and lipoprotein changes in African subjects.
Guinea pigs were sensitized intracutaneously for 7 days (0.1 ml of 1% aldehyde solution/day) with nine aldehydes (p-anisaldehyde, benzaldehyde, citral, ethylvanillin, furfural, n-octanal, l-perillaldehyde, piperonal and vanillin) which are used as the food flavoring agents. Three weeks later, each aldehyde was injected by different concentrations (0.25-1.0%). Skin reactions were observed 24 hr after the injection of the aldehydes. All aldehydes tested exhibited a positive skin reaction, indicating that these aldehydes are capable of inducing an allergic reaction.