8-Hydroxydeoxyguanosine in DNA from TPA-stimulated human granulocytes.

8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH.) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH. is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH.) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human granulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8-OHdG/10(5) dG).

Regeneration of vitamin E in human platelets.

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications.

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.

Reproduction in Fischer-344 rats exposed to methyl chloride by inhalation for two generations.

Male and female Fischer-344 rats were exposed to methyl chloride by inhalation (0, 150, 475, or 1500 ppm, 6 hr/day, 5 days/week, 40 males and 80 females per group). The only treatment-related clinical signs were a 10 to 20% body weight gain depression (BWGD) in both males and females exposed to 1500 ppm at all weekly weighings after 2 weeks of exposure and a 5-7% BWGD in 475-ppm exposed animals after Day 57. After 10 weeks the exposure schedule was changed to 6 hr/day, 7 days/week and each male was mated to two exposed females. The mating period was ended after 2 weeks, at which point 10 males/group were necropsied. The only treatment-related lesions found were severe bilateral testicular degeneration (10/10) and granulomas in the epididymis (3/10) in the 1500-ppm males. The remaining 30 males per group were then removed from exposure and mated during a 2-week period with 60 unexposed females. The exposed females were continued on exposure from the start of mating to Postnatal Day 28 (6 hr/day, 7 days/week). The females were not exposed from Gestation Day 18 to Postnatal Day 4, and the pups were never directly exposed prior to weaning. There were no significant differences between groups in the number of exposed or unexposed females that mated, as evidenced by copulation plugs. No litters were born to exposed or unexposed females mated to the 1500-ppm males. There was no significant difference in the number of litters produced by the 150-ppm groups when compared to the control groups. Fewer litters were born in the 475-ppm groups than in the control groups. No differences in litter size, sex ratio, pup viability, or pup growth were found among the 475-ppm, 150-ppm, or control F0 groups. When bred 10 weeks after the cessation of exposures, 5 to 20 1500-ppm F0 males had regained the ability to sire normal litters. The same number of 475-ppm F0 males proved as fertile (15/20) as control F0 males (13/20). After weaning, F1 pups from the 475-, 150-, and 0-ppm groups were exposed to the same concentrations of methyl chloride for 10 weeks and then mated. A trend toward decreased fertility was found in the 475-ppm F1 group.

An evaluation of a water purification system for use in animal facilities.

A commercially available water purification system was evaluated for its ability to minimize chemical and microbial contaminants. The reduction or removal of these impurities from the drinking water of experimental animals would reduce experimental variability. 3 strains of bacteria were collected from the processed water. An increase in the total number of bacteria was observed the longer the filters remained in use. Determinations of heavy metals in water samples before and after processing were made for lead, zinc, copper, nickel, manganese, iron, arsenic and mercury. Calcium and magnesium levels were also determined. The concentrations of these inorganic chemicals were reduced by the purification process except at 2 time points in which desorption of the chemical could have occurred. Bacterial colonization and desorption of these chemicals were controlled by installing new filter cartridges. Volatile halocarbon concentrations were determined for water samples before and after purification. All volatile halocarbons analyzed were less than 10 ppb before and after purification at all time points. Other organic chemicals were greatly reduced by the purification process. In a study of contaminants associated with installation of the unit, it was found that flushing the unit for 8 days reduced lead and methyl ethyl ketone concentrations to insignificant levels. The purification system was found to be effective in providing high quality drinking water as verified by a microbial and chemical testing program.

A water valve hole coverplate for reducing traumatic death in mice.

Mice often attempted to escape through the opening for an automatic water valve when hanging wire cages were opened. Traumatic death or injury resulted when the cage was closed. To minimize these deaths and injuries, a coverplate was designed and tested. The coverplate prevented mice from escaping through the water valve hole during times when the cage was open and eliminated traumatic deaths or injury from being crushed by the valve as the cage was closed.

Differences in the microenvironment of a polycarbonate caging system: bedding vs raised wire floors.

The microenvironment of polycarbonate cages housing rats with and without various types of bedding was compared with that of cages that utilized wire floor inserts with different bedding types. Parameters monitored were temperature, humidity, ammonia concentrations and particulates. No differences were noted in the various caging types in relation to temperature and humidity measurements. Significant differences in ammonia concentrations existed in some of the cages when bedding material was used. The use of raised floorwalk inserts also demonstrated significant differences in particulate counts to cages without inserts. The data obtained demonstrated that contact bedding was useful in controlling ammonia generation and that a raised floorwalk insert reduced significantly the aerosolization of bedding particles that could be ingested or inhaled by the rats.