Hideki Kurita

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.

The changes in immune response of guinea pigs intrapleurally injected once with beryllium oxide (BeO) or beryllium copper alloy (Be-Cu) were examined. To evaluate the changes in immune response of guinea pigs, two indicators, beryllium lymphocyte transformation test (Be-LTT) and complement hemolytic activity (CH50), were used. The BeO or Be-Cu injection groups was divided three groups, a high concentration injection group (36 mu g Be dose), a low concentration injection group (3.6 mu g Be dose) and the control group. The Be-LTT and CH50 were determined in the blood of guinea pigs at 4, 8 and 16 weeks after Be administration. The Be-LTT value at the 4th week in the BeO groups (a high or low concentration injection group, respectively) showed a statistically significant increase (p<0.01) compared with that of the control. In the Be-LTT value for BeO at 8 and 16 weeks after Be injection and the Be-Cu groups, there were no statistical difference between the Be administration groups and the control. On the other hand, only the CH50 of the high concentration group at the 8th week in the BeO injection group was significantly decreased (p<0.01) compared with that of the control. In the other Be injection group, no significant changes in the CH50 was observed compared with the control group. On the basis of the results observed in the present study, there may be a slight difference in the effect on the immune response of guinea pigs caused by BeO or Be-Cu administration. The immunotoxicity of Be in guinea pigs may manifest itself more intensely in the cellular immune response than in the humoral immune response.

On the basis of our clinical experience in treating bronchial asthma that occurred due to chloroplatinate among manufacturers of oxygen sensors, we developed an intratracheal inhalation-exposure apparatus for small animals for the purpose of explicating the mechanism of allergic development of this disease and conducted an experimental study on acute toxicity due to an inhalation-exposure to platinum chloride acid (H2PtCl.6H(2)O) in guinea pigs. The results of the respiratory function tests showed that after the inhalation of platinum chloride acid, the Paw (airway pressure) and Pr (pulmonary resistance) levels increased and Cdyn (dynamic compliance) decreased in a manner dependent on the concentration of inhaled platinum compounds. The appearance of a peak of the airway reactions to the platinum chloride inhalation-exposure was delayed when antihistaminics had been administered compared with the corresponding reactions of the nonantihistaminics-administered group. A significant correlation was noted between the Paw increase rate and histamine levels in BALF and plasma (p<0.05).

The relationship between the beryllium (Be) level in the working environment and the beryllium lymphocyte transformation test (Be-LTT) value was studied on the basis of data in the survey of the 5 years from 1992 to 1996. The subjects were two working shops, beryllium-copper (Be-Cu) alloy manufacture and Be-Cu metal mold manufacture. The results showed that the higher the Be levels in working environments become, the more the Be-LTT values of workers are enhanced. Thus, it was made clear that changes in the Be-LTT values of workers were related to the Be levels in working environments. We emphasize that it is important to regularly check the Be levels in working environments, as well as the Be-LTT values of workers to prevent the development of chronic beryllium disease.

The relationship between airborne concentration of beryllium in the working environment and workers' beryllium lymphocyte transformation test (Be-LTT) values was examined based on data obtained from a four-year survey (1992-1995) conducted at beryllium-copper alloy manufacturing factories. This study showed that the T cells of workers continuously exposed to beryllium of more than 0.01 mu g/m(3) could be activated and that the cell-mediated immune response of workers could be promoted. On the other hand, the Be-LTT of workers exposed to beryllium levels of less than 0.01 mu g/m(3) was shown to be unaffected by beryllium. These findings suggest that beryllium sensitization is not manifested when level of beryllium in working environment are less than 0.01 mu g/m(3). Therefore, in such cases workers do not develop Chronic beryllium disease (CBD). We concluded that the Be-LTT can be applied as a medical indicator to detect the development of CBD.

Immunocytotoxicity of beryllium (Be) was evaluated by studying cell viability, intracellular DNA synthesis and SRBC-IgM response in an in-vitro culture system using non-sensitized spleen cells of a C57BL mouse. Be addition showed a suppressive effect on cell viability, an enhancing effect on DNA synthesis and on IgM antibody production. The suppressive effect on cell viability manifested itself markedly as the concentration of Be was increased or the culture time was prolonged. The DNA synthesis-enhancing effect was noted at a relatively low concentration of Be (not more than 10μM). The enhancing effect on the IgM response was related to Be concentration at not more than 20μM. The experimental results mentioned above speculate that the cytotoxicity of Be shows a conflicting pattern of enhancement or suppression according to the concentration used and that immunologically it has a modulating effect or an activating effect on the immunocompetent cells.

Placental transfer and body distribution of triphenyl tin chloride (TPTC) and tin chloride (SnCl2) were investigated in pregnant rats. Pregnant Wistar rats were orally administered TPTC at 0, 1.0 and 2.0 mg/rat/day during 6 ~ 10 and 14 ~ 18 days of gestation, and with SnCl2 at 0 and 20 mg/rat/day on 7, 10 and 15 days of gestation (group treated 3 times) or 6 ~ 10 and 13 ~ 16 days of gestation at 0 and 20 mg/rat/day (group treated 9 times). On day 20 of gestation in rats treated with TPTC, and day 17 in rats with SnCl2, fetuses, placentas, maternal blood and organs were obtained for analyses. Mean fetal tin levels from rats treated with 1.0 and 2.0 mg TPTC were 0.05 and 0.11 μg/g wet weight, respectively, these values being significantly (P &lt 0.01) higher than in the control group. The latter was higher than the former at a significance level of 5%. No significant difference between fetal tin levels from rats treated with SnCl2 and the control group could be detected. Following oral TPTC administration, tin was present in maternal liver, kidney and brain in high concentrations. Following the oral administration of SnCl2, the tin level was highest in bone and kidney, but none could be detected in the brain.