Search

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

Platelet satellitosis of polymorphonuclear cells is a phenomenon induced or enhanced by the anticoagulant EDTA. In contrast with previously reported studies, the subject in the present case did not demonstrate platelet satellitism but was profoundly pseudothrombocytopenic owing to platelet phagocytosis. Virtually all polymorphonuclear leukocytes and monocytes contained numerous ingested platelets in contrast with previous cases in which phagocytosis was observed only rarely and involved ingestion of single cells. The phenomenon was documented by immunocytochemical staining and transmission electron microscopy. Autoantibodies were detected in EDTA-anticoagulated blood. However, neither platelet antibody nor phagocytosis was present when heparin, acid-citrate dextrose, or citrate was used as an alternative anticoagulant. The antibody was not temperature dependent. Mixing studies showed the transfer of the phagocytosis phenomenon to healthy donors. Although platelet function assays are typically normal in EDTA-dependent platelet satellitism, this subject showed no secondary aggregation wave in response to adenosine diphosphate and depressed adenosine triphosphate release with collagen, adenosine diphosphate, and arachidonic acid.

Subacute Toxicity of a Purine Nucleoside Phosphorylase Inhibitor in Rats. Wolfgang, G. H. I., Bleavins, M. R., Hallak, H., Kasali, O. B., and Urda, E. (1995). Fundam. Appl. Toxicol. 28, 51-58. Rats received daily oral doses of 15, 50, 150, or 200 mg/kg CI-1000 for 4 weeks. Doses were selected based on findings from a 2-week range-finding study where doses of 250 and 500 mg/kg resulted in mortality. In the 4-week study, females given 200 mg/ kg were sacrificed during Week 2 due to poor condition. Serum creatinine and urea nitrogen increased 2- to 2.5-fold in females given 200 mg/kg. Dose-related increases in urine volume, urinary protein excretion, and osmolar excretion occurred in both sexes beginning at 50 mg/kg. Kidney weights increased 9-40% in both sexes at ⩾50 mg/kg; histopathologic changes were confined to the 150 and 200 mg/kg groups. At Week 4, T-suppressor/cytotoxic lymphocytes were reduced 43% and T-helper/inducer lymphocytes were reduced 22% in males given 200 mg/kg. In females, T-sup-pressor/cytotoxic lymphocytes were significantly decreased (approximately 40%) at 50 and 150 mg/kg, with no significant effects on T-helper/inducer lymphocyte populations. At Week 8, following 4 weeks without treatment, T-lymphocyte subpopulations were similar in control and drug-treated groups. B-lymphocyte counts and percentages were increased at Weeks 4 and 8 in males receiving 150 or 900 mg/kg. Thymic weights decreased at Week 4 at doses of 150 and 200 mg/kg. Plasma CI-1000 levels were higher in females than in males at all doses except 15 mg/kg; Cmax and AUC values were largely dose proportional in both sexes. In summary, CI-1000 was well-tolerated at doses of 15, 50, and 150 mg/ kg with no adverse effects occurring at 15 mg/kg. Drug-induced changes in the kidney were mild and reversible. Immunomodulatory effects were noted at doses of 50 mg/kg or higher.

The microsomal enzyme cytochrome P450 2D6 (CYP2D6), also known as debrisoquine 4-hydroxylase, is involved in the oxidative metabolism of many widely used drugs, including neuroleptics, tricyclic antidepressants, antiarrhythmics, and β-adrenergic blocking agents (1). Polymorphisms of CYP2D6 are the best characterized examples of genetically mediated effects on a drug-metabolizing enzyme of clinical importance (2). When a drug that is a CYP2D6 substrate is taken by different individuals, it is not uncommon to observe large differences in plasma concentrations at steady state. This is explained, in part, by the three clinically distinct phenotypes associated with the CYP2D6 gene, normal metabolizers, poor metabolizers, and rapid metabolizers. In normal metabolizers, steady-state plasma drug concentrations fall within the desired therapeutic range and toxic effects are nonexistent or minimal. In fast-metabolizer individuals, desired concentrations are below therapeutic, and these patients generally do not respond at the recommended dosing regimen. In poor-metabolizer individuals, drug concentrations are above therapeutic level and undesired toxicity can be evoked.

High-density microarrays are useful tools to study gene expression for the purpose of characterizing functional tissue changes in response to the action of drugs and chemicals. To test whether high-density expression data can identify mechanisms of toxicity and to identify an unknown sample through its RNA expression pattern, groups of male Wistar rats were administered 6 hepatotoxicants. The compounds chosen for this study were microcystin-LR (MLR), phenobarbital (PB), lipopolysaccharide (LPS), carbon tetrachloride (CT), thioacetamide (THA), and cyproterone acetate (CPA). These hepatotoxicants are known to induce adverse liver effects through different mechanisms. Liver mRNA was isolated and used to generate biotinylated cRNA for hybridization to a custom 1,600–rat gene DNA microarray. Treatment correlation matrices analyzed hybridization data from a hepatotoxicant-blinded sample, with gene expression coefficients (GEC) evaluated by means of hierarchical cluster analysis and visual representation as dendrograms. The experimental liver toxicity from the different treatments was confirmed by means of concurrent histopathology, liver enzymes, and bilirubin assays. This toxico genomic analysis identified multiple genes and groups of genes that were affected by the hepatotoxicants on study, indicating that high-density microarray expression data are useful to identify groups of genes involved in toxicity. In addition, the mRNA expression profile of an unidentified sample can be accurately identified when compared with the expression profiles resident in the data set. This study supports the use of gene expression–profiling technology to determine or to predict toxic liver effects. (HEPATOLOGY 2001;33:1239-1258.)

AIMS: The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. METHODS: A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. RESULTS: This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. CONCLUSIONS: This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.

CI-1000, a 9-deazaguanine analog, is an orally active and reversible inhibitor of purine nucleoside Phosphorylase (PNP). In vitro, CI-1000 inhibits human T-lymphoblast replication and the mixed lymphocyte response, as well as facilitating the intracellular accumulation of deoxyguanosine triphosphate (Gilbertsen et al., 1992; Dong and Gilbertsen, 1993; Wilburn et al. 1993). These changes are consistent with inhibition of PNP (Osborne and Scott, 1983; Fairbanks et al., 1990; Gilbertsen and Sircar, 1990) and the drug is approximately 10-fold more potent than a structurally related compound (Gilbertsen et al., 1991a, 1991b, and 1992).

Feeding adult mink and European ferrets diets that contained 1, 5, or 25 mg/kg added hexachlorobenzene (HCB) resulted in reduced reproductive performance as indicated by decreased litter size, increased percentage of stillbirths, increased kit mortality, and decreased early kit growth. Diets treated with 125 or 625 mg/kg HCB were lethal to the adults of both species. In general, the mink were more sensitive to the toxic effects of HCB than were ferrets.

Adult female ferrets were found to absorb 85.4% of a single dietary dose of the polychlorinated biphenyl (PCB) mixture Aroclor 1254. Excretion in the days immediately following dosing yielded the greatest quantity of PCBs eliminated in a given time period. In general, urinary excretion represented one-tenth or less of the PCBs excreted via the feces. Placental transfer to the ferret kits was 0.01% (per kit) of the female's absorbed dose when exposure occurred during the first trimester of pregnancy and 0.04% (per kit) when the PCBs were administered during the third trimester. Transplacental exposure to PCBs was considerably less than that reaching the kits through the dam's milk. The ratio of placental to mammary transfer, following 1 week of lactation, was calculated to be approximately 1:15 for the offspring of first-trimester-dosed females and 1:7 for the offspring produced by females treated during the third trimester.

Adult female standard dark mink were exposed to hexachlorobenzene (HCB) at concentrations of 0, 1, and 5 ppm in the feed and bred with males on the same treatments. Female offspring were allowed to mature to 16–17 weeks and killed. At 16–17 weeks of age, HCB had no effect on body weights or liver weights. Hepatic cytochrome P-450 and ethoxyresorufin-O-deethylase were significantly increased 2.0- and 1.5-fold, respectively, in the 5-ppm treatment group. Electron microscopy failed to reveal proliferation of the smooth endoplasmic reticulum. No hepatic damage was observed. No changes in in vitro renal function, measured as accumulation of para-aminohippurate and tetraethylammonium by renal cortical slices, were detected in any treatment group. Histological examination of renal slices did not reveal any alterations in morphology. Fat was the predominate site of HCB disposition; samples from the 5-ppm treatment group contained 626.10 ± 12.01 ng HCB/g tissue. Whereas perinatal HCB administration has profound effects on the survival of off-spring born to exposed mink, only induction of hepatic mixed-function oxidases was observed in the surviving kits without any observable frank hepatotoxicity.

Hexachlorobenzene (HCB) was found to depress the lymphocyte blastogenic response of mink (Mustela vison) and European ferrets (Mustek putorius furo) to the plant lectin Concanavalin A (Con-A). A significant reduction in the incorporation of [3H]thymidine was seen in the lymphocytes from adult animals consuming 25 ppm dietary HCB for 8 months. This effect was observed in animals showing no physical signs of distress or toxicity. The in utero and early postnatal exposure of young mink to HCB also resulted in a significant depression of lymphocyte responsiveness. Exposure levels as low as 1 ppm in the dam's diet caused an impaired T-cell response. In the young ferrets exposed to HCB in utero and during nursing, no change in the stimulation index was seen. The monitoring of immunocompetence in animals exposed to toxicants, either through the diet or indirectly via their dam, provides a sensitive indicator of toxicity. Effects were seen in activation of lymphocytes from the mink and ferrets used in this study without grossly observable clinical signs being present.

The effects of 3,3′,4,4′-tetrachloroazoxy-benzene (TCAOB) on the immune system of mice was examined and compared with cyclophosphamide (CY). This chemical can be produced as a result of microbial degradation of commonly used chloroaniline herbicides. Herbicides of the acylan-iline class generally have a low mammalian toxicity while remaining relatively inexpensive. Therefore, TCAOB could be formed anywhere in the environment where choroaniline pesticides are used. TCAOB treatment caused thymic atrophy and a decrease in white blood cell (WBC) count after sheep red blood cell (SRBC) immunization. A decline in the number of Lyt-1+ cells (T-helper lymphocytes) was seen in all TCAOB animals, with unimmunized mice also showing a decreased number of Lyt-2.2+ cells (T-suppressor lymphocytes). No change was found in the ratio of these two cell types. TCAOB did, however, result in a severe reduction in the number of plaque-forming-cells (PFCs) and in serum antibody concentration. CY caused a decrease in thymus weight, WBC count, the number of cells recovered per spleen, and the relative percentages of Lyt-l+ and Lyt-2.2+ cells recovered. The mice exhibited a lower lymphocyte blastogenic response to lipopolysaccharide (LPS) than control animals, but no change in Concanavalin A (Con-A) responsiveness. CY also resulted in a severe drop in the number of PFCs and the quantity of antibody produced following SRBC immunization.

Female European ferrets (Mustela putorius furo) absorbed 98.5% of a single dietary exposure of hexachlorobenzene (HCB). The HCB was found to readily cross the placenta and to be excreted in the milk of pregnant/lactating ferrets. After consuming HCB-treated feed, ferrets raising offspring excreted 50% of the initial dose by 32 d, while unbred ferrets achieved this same degree of HCB elimination in 41 d. The percentages of HCB excreted via the urine and feces were approximately 5 and 45%, respectively, in both groups at the 50% stage of elimination. Adipose tissue was the most significant long-term repository for HCB in the ferret. The other tissues analyzed for [14C]HCB showed a general relationship of increased radioactivity with increased fat content of the tissue. The ferrets with nursing kits were able to significantly reduce their body burden of HCB when compared to unbred females. The developing ferret kits were subjected to HCB insult both in utero and via dam's milk. The ratio of milk to placental exposure in the growing offspring was calculated to be 31:1. Thus, in addition to any toxic effects HCB may have on the adult reproducing population, the placental and mammary transfer of HCB constitutes a potential threat to the developing and growing animal.

After a single dose of [14C]hexachlorobenzene (HCB) via gavage into the crop, the accumulation of [14C]HCB in female bobwhite (Colinus virginianus) tissues occurred to the greatest extent in adipose tissue followed by skin, liver, brain, heart and kidney, whole blood, and muscle. There was a general relation between increasing HCB concentration and increasing fat content of the tissue. Absorption of [14C]-HCB was rapid with peak radioactivity occurring at 4 h in all tissues except for fat and skin, where it continued to rise until 32 and 16 h after dosing, respectively. Elimination of HCB from tissue was biphasic with phase I representing the combination of HCB excretion and HCB redistribution from tissue into fat stores. Phase II represented solely HCB excretion, which appeared to be a first-order process. The half-life of [14C]HCB in tissues, feces, and eggs ranged from 9-13 d regardless of HCB concentration. Radioactive HCB accumulation in egg yolk was a significant mechanism for the removal of this chemical from bobwhite and accounted for 50% of the total HCB excreted.

In the initial trial, adult mink and ferrets were administered hexachlorobenzene (HCB) via the feed at concentrations of 1, 5, or 25 ppm for 47 wk. Animals receiving 125 and 625 ppm HCB in the diet died before termination of the experiment, with female ferrets at the 125 ppm level displaying abnormal aggressiveness and hyperexcitability just prior to death. Hypothalamic serotonin (5-HT) was significantly elevated at all dose levels in mink, and cerebellar 5-HT was significantly elevated at 1 ppm in the ferret. Regional brain biogenic amine concentrations were also determined in the offspring of the female mink that were administered 1 and 5 ppm HCB. Hypothalamic dopamine (DA) concentrations were significantly depressed by 1 and 5 ppm in these kits. In a second study, adult male and female ferrets were administered 250 or 500 ppm HCB via the diet for 7 wk. Two animals at the 250-ppm level and 3 animals at the 500-ppm level died before termination of the experiment without showing behavioral changes. Of the remaining animals, 3 ferrets at 250 ppm and 1 ferret at 500 ppm showed slight aggressiveness and hyperexcitability during the last week of the experiment. Concentrations of 5-HT were significantly elevated at 500 ppm in the cerebral hemispheres and at 250 ppm in the midbrain of male ferrets, while in the females, 5-HT was elevated in the cerebral hemispheres at 250 ppm and in the hypothalamus at both 250 and 500 ppm. Norepinephrine (NE) concentrations were significantly elevated in the cerebellum of males exposed to 250 and 500 ppm, as were NE concentrations in the midbrain. HCB at 500 ppm caused a significant increase in medullary NE, while 250 ppm caused an increase in hypothalamic NE in males. The only change in regional brain dopamine (DA) concentrations occurred at 500 ppm HCB in the midbrain of males, where there was a significant elevation of this neurotransmitter.