Toxicity and potential carcinogenicity studies of boric acid were investigated in mice to verify in a second rodent species that this was a noncarcinogenic chemical. Earlier chronic studies in rats indicated boric acid was not a carcinogen. The chemical is nominated for testing because over 200 tons are produced annually, there are multiple uses for the product, and there is potential for widespread human exposure, both orally and dermally. Both sexes of B6C3F1 mice were offered diets mixed with boric acid for 14 days, 13 weeks, or 2 years. Dietary doses used in the acute, 14-day study were 0, 0.62, 1.25, 2.5, 5, and 10%; those in the subchronic, 13-week study were 0, 0.12, 0.25, 0.50, 1, and 2%; and doses in the 2-year, chronic study were 0, 0.25, and 0.50% in the diet. Mortality, clinical signs of toxicity, estimates of food consumption, body weight gain, and histopathologic examination of selected tissues constituted the variables measured. In the 14-day study mortality was proportional to dose and time of exposure in both sexes, occurring in dose groups as low as 2.5% and as early as 7 days of exposure. Body weights were depressed more than 10% below controls in the higher dose groups of both sexes. Mortality in the 13-week study was confined to the two highest dose groups in male mice and to the 2%-dose group in females. Body weight depression from 8 to 23% below those of controls occurred in the 0.50% and higher dose groups of both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

The toxicity of cinnamaldehyde (CNMA) was compared after administration by gavage and in dosed feed. Rats and mice of both sexes received CNMA by daily corn oil gavage (for 2 wk), or in microencapsulated form in feed (2 wk for rats, 3 wk for mice). Feed formulations contained 0–10% CNMA microcapsules, equivalent to approximate daily doses of 0–3000 mg CNMA/kg body weight for rats and 0–10,000 mg CNMA/kg body weight for mice. Concentrations were chosen to deliver CNMA doses approximately equal to doses in the gavage study. Gavage doses of 2620 mg/kg/day and above in mice and 940 mg/kg/day and above in rats produced nearly 100% mortality; there were no deaths in animals receiving microencapsulated CNMA. Rats and mice receiving CNMA in feed showed a dose-related decrease in body weight gain, which was accompanied in rats by hypoplastic changes in reproductive organs and accessory sex glands. CNMA administration by either route caused hyperplasia of the forestomach mucosa. These results demonstrate that microencapsulation in feed can present a useful alternative to gavage dosing for repeated-dose or prolonged-exposure studies, in that (1) the toxic effects of CNMA were similar after gavage dosing and after administration in microencapsulated form in feed, (2) ingestion of chemical in the feed more closely approximates human exposures, and (3) microencapsulation allows the delivery of higher net doses of chemical, while avoiding the acutely toxic effects of a bolus dose.

A study of the potential effects of microencapsulation on the toxicity of citral was conducted in 14-day continuous feeding studies with both sexes of F344 rats and B6C3F1 mice. Toxicity by the feeding route was compared with that from bolus doses of the neat chemical in corn oil administered by gavage. Both sexes of rats and mice were given diet containing 0, 0.63, 1.25, 2.5, 5 and 10% citral microcapsules. These feed formulations were equivalent to daily doses of 0, 142, 285, 570, 1140 and 2280 mg citral/kg body weight for rats and 0, 534, 1068, 2137, 4275 and 8550 mg citral/kg body weight for mice. The daily gavage doses were 0, 570, 1140 and 2280 mg citral/kg body weight for both sexes of rats, and 0, 534, 1068 and 2137 mg citral/kg body weight for both sexes of mice. Citral microcapsules administered in the diet did not cause mortality in mice or rats. Toxicity was confined to decreases in body weight at the 10% concentration in mice, at the 5 and 10% concentrations in rats, and decreases in absolute weights of the liver, kidney and spleen at the 10% concentration in rats. The only histopathological change observed was minimal to mild hyperplasia and/or squamous metaplasia of the respiratory epithelium in the anterior portion of the nasal passages of rats fed 5 or 10% citral microcapsules. By contrast, citral gavage caused mortality in five out of five male and female mice at 2137 mg/kg body weight, and in two out of five male mice at 1068 mg/kg body weight. There were dose-related increases in absolute liver weights of male and female mice. Cytoplasmic vacuolization of hepatocytes occurred in all female mice gavaged with 1068 and 2137 mg citral/kg body weight, and in male mice from the 2137 mg/kg dose group. Necrosis, ulceration and/or acute inflammation of the forestomach occurred in the high-dose mice of both sexes. Inflammation and/or hyperplasia of the forestomach occurred in about half of the male and female mice dosed with 1068 mg citral/kg. Citral gavage at doses that were equivalent to up to 10% in the diet (2280 mg/kg body weight) did not cause toxicity in rats, except for minimal hyperplasia of the squamous epithelium of the forestomach in high-dose males. Microencapsulated citral given in the diet has distinct advantages as an alternative route of administration for long-term studies: the microencapsulation process prevents the normally rapid degradation of the chemical; continual ingestion from the diet mimics the human route of exposure; and higher doses of the chemical can be attained in the animal model to maximize the detection of potential toxic or carcinogenic responses.

An animal model using rats was developed to initiate investigations on the bioavailability of different sources of environmental lead. Lead must be absorbed and transported to target organs like brain, liver, kidney, and bone, before susceptible cells can be harmed. The bioavailability and therefore the toxicity of lead are dependent upon the route of exposure, dose, chemical structure, solubility, particle size, matrix incorporation, and other physiological and physicochemical factors. In the present study male F344 rats were fed < or = 38 microns size particles of lead sulfide, lead oxide, lead acetate, and a lead ore concentrate from Skagway, Alaska, mixed into the diet at doses of 0, 10, 30, and 100 ppm as lead for 30 d. No mortality or overt symptoms of lead toxicity were observed during the course of the study. Maximum blood lead concentrations attained in the 100 ppm groups were approximately 80 micrograms/dl in rats fed lead acetate and lead oxide, and were approximately 10 micrograms/dl in those fed lead sulfide and lead ore concentrate. Maximum bone lead levels in rats fed soluble lead oxide and lead acetate were much higher (approximately 200 micrograms/g) than those seen in rats fed the less soluble lead sulfide and lead ore (approximately 10 micrograms); kidney lead concentrations were also about 10-fold greater in rats fed the more soluble compared to the less soluble lead compounds. However, strong correlations between dose and tissue lead concentrations were observed in rats fed each of the four different lead compounds. Kidney lesions graded as minimal occurred in 7/10 rats fed 30 ppm and in 10/10 rats fed 100 ppm lead acetate, but not at lower doses or from other lead compounds. Similarly, urinary aminolevulinic acid excretion, a biomarker for lead toxicity, was increased in rats fed 100 ppm lead acetate or lead oxide, but was unaffected at lower doses or by the less soluble lead compounds. Although the histological and biochemical responses to lead toxicity were restricted to the more soluble lead compounds in this study, lead from Skagway lead ore concentrate and lead sulfide was also bioavailable, and accumulated in proportion to dose in vulnerable target organs such as bone and kidney. Longer-term studies with different mining materials are being conducted to determine if tissue lead continues to increase, and whether the levels attained are toxic. Data from such studies can be used to compare the toxicity and bioavailability of lead from different sources in the environment.

Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile compound used as a chemical intermediate, a catalyst, and an antiknock additive in gasoline. This organometallic chemical is of particular interest because of its structural similarities to other metallocenes, some of which are carcinogenic. F344/N rats and B6C3F1 mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m3, 6 hr/day, 5 days/week, for 13 weeks. During these exposures, no rats or mice died, nor were any clinical signs of ferrocene-related toxicity observed. At the end of the exposures, male rats exposed to the lowest and highest level of ferrocene had decreased body weight gains compared to filtered-air-exposed control male rats, while body weight gains for all groups of both ferrocene- and filtered-air-exposed female rats were similar. Male mice exposed to ferrocene had no differences in body weight gains, compared to controls, but female mice had decreases in body weight gains at the 10 and 30 mg/m3 exposure levels. There were exposure concentration- and exposure-time-related increases in lung burdens of iron. The mean iron lung burden in rats exposed to 30 mg ferrocene vapor/m3 for 90 days was four times greater than the burden in control rats. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen, clinical chemistry, and hematology parameters were observed. This suggests that the accumulations of iron in lung did not cause an inflammatory response nor any functional impairment of the lung. There were no indications of developing pulmonary fibrosis nor of any hematologic toxicity. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Exposure-related histopathologic alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium where pigment accumulation, necrotizing inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. Histochemical stains of these target tissues showed the presence of iron ions. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalyzed lipid peroxidation of cellular membranes or the iron-catalyzed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA.

Sixty-day bioassays of iodinated glycerol, trichlorfon, and acetaminophen were conducted using a leukemia transplant model in 6- to 8-week-old F344 rats to investigate the potential of these chemicals to affect tumor progression. The chemicals were administered in the drinking water at doses that approximated those used in previously conducted 2-year carcinogenesis studies. Simultaneous with dose administration, half of a group of young, healthy, syngeneic rats were given subcutaneous transplants of mononuclear cells derived from spleens of leukemic donors. Variables used to quantitate tumor progression included body weight, spleen weight, white blood cell (WBC) and red blood cell (RBC) counts, packed cell volume, hemoglobin concentration, and platelet counts. Iodinated glycerol at 1.25 or 2.5 mg/ml caused a greater increase in leukocytosis in dosed transplant recipients in comparison to that experienced by undosed recipients: trichlorfon at 2.5 or 5.0 mg/ml enhanced splenomegaly and induced greater reductions in RBC parameters in dosed recipients in comparison to that experienced by undosed recipients. Acetaminophen at 3.0 and 6.0 mg/ml resulted in insignificant but dose-related increases in spleen weight and leukocytosis only in the female rat transplant recipients, as was observed in 2-year studies. Based on results from the short-term leukemia transplant model, data from 2-year carcinogenicity studies, and structure-activity considerations, exposure to iodinated glycerol and trichlorfon was more strongly associated with the expression of leukemia than exposure to acetaminophen. The potential carcinogenicity of each of these chemicals should be taken into consideration when calculating estimates of risk and decisions for their use.

The toxicokinetic profile of cinnamaldehyde (CNMA) was investigated in Fischer 344 rats. CNMA was found to be unstable in blood. After iv administration, a large fraction of CNMA was immediately oxidized to cinnamic acid. The biological half-life of CNMA after iv administration was found to be 1.7 hr. After administration by gavage of CNMA at 250 or 500 mg/kg body weight using corn oil as vehicle, the maximum blood concentrations of CNMA were in the order of 1 μg/ml. These low blood concentrations were maintained over a 24-hr period after a dose of 500 mg/kg, which is relatively long considering the short (1.7 hr) biological half-life of CNMA. The estimated oral bioavailability of CNMA was less than 20% for both the 250 and 500 mg/kg doses. No CNMA was present in blood at any time in rats dosed with 50 mg CNMA/kg body weight. Only a small amount of the administered CNMA was excreted in rat urine as free cinnamic acid or β-glucuronide-conjugated cinnamic acid. The majority of CNMA administered orallywas excreted in urine as hippuric acid within 24 hr. The maximum excretion rate occurred at 8 hr after gavage. Hippuric acid recovered in 50-hr urine samples was found to be directly proportional to the oral dose of CNMA.

Both sexes of F344 rats were gavaged with maximal tolerated doses of mercuric chloride for periods from 2 wk to up to 2 yr to investigate chronic nephrotoxicity and potential carcinogenicity. The toxicity of mercuric chloride was excessive after 2 wk of exposure to doses ranging from 1.25 to 20 mg/kg, compromising renal function by selectively destroying cells of the proximal tubules, and eliciting marked elevations in urinary biomarker enzymes diagnostic for acute renal tubule necrosis. In the 2-wk studies, urinary alkaline phosphatase and aspartate aminotransferase were most sensitive to renal mercury toxicity among a panel of six enzymes, exhibiting twofold increases above controls at the 5.0 mg/kg dose, before changes in the other enzymes occurred. Urinary lactate dehydrogenase was the most responsive enzyme, with up to 11-fold increases in activity above controls. In response to mercuric chloride exposure of 5.0 mg/kg for 2-6 mo, the greatest and most persistent increases in elevation of urinary enzyme activities were exhibited by alkaline phosphatase and gamma-glutamyl transferase, which increased two- to threefold above controls. At this interval, the maximal severity of the renal lesions in both sexes of rats was graded as minimal to mild. Beyond 6 mo none of the urinary enzymes measured in this study was adequate as biomarkers of nephrotoxicity, although the severity of the renal lesions had progressed. Mercury accumulated in a dose-related fashion primarily in the kidney, and to a lesser extent in the liver The severity of the renal lesions was increased by continued exposure to mercuric chloride, as tissue concentrations of mercury rose in proportion to dose. Mercuric chloride treatment for 2 yr clearly exacerbated the severity of the spontaneous nephrotoxicity prevalent in aging F344 rats. The excessive mortality that occurred in the male rats was probably due to a combination of these factors. No renal tumors were detected in rats, possibly because the potential for their development was reduced; however direct tissue contact with mercury induced squamous-cell papillomas of the forestomach in both sexes.

A rapid and sensitive high performance liquid chromatographic (HPLC) method is described for the quantitation of cinnamaldehyde (CNMA) in rat blood at concentrations of 0.1-100 micrograms/mL. One of the metabolites of CNMA, cinnamic acid, can also be quantified simultaneously. CNMA is unstable in rat blood, probably because of rapid oxidation to cinnamic acid by enzymatic catalysis and nonenzymatic Schiff base formation with free amine groups of blood proteins. The disappearance of CNMA from rat blood follows first-order reaction kinetics with a half-life of 9 min at room temperature. The current analysis method involves the addition of an agent that will prevent CNMA degradation by denaturing protein and competitively blocking nucleophilic addition reactions, resulting in the nearly complete recovery of CNMA from blood. Recovery of cinnamic acid was approximately 80% at concentrations of 1-10 micrograms/mL.

Two-Week, Repeated Inhalation Exposure of F344/N Rats and B6C3F1 Mice to Ferrocene. SUN, J. D., DAHL, A. R., GILLETT, N. A., BARR, E. B., CREWS, M. L., EIDSON, A. F., BECHTOLD, W. E., BURT, D. G., DIETER, M. P., AND HOBBS, C. H. (1991). Fundam. ApplToxicol. 17, 150-158. Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile, organometallic compound used as a chemical intermediate, a catalyst, and as an antiknock additive in gasoline. It is of particular interest because of its structural similarities to other metallocenes that have been shown to be carcinogenic. F344/N rats and B6C3F, mice were exposed to 0, 2.5, 5.0, 10, 20, and 40 mg ferrocene vapor/m3, 6 hr/day for 2 weeks. During these exposures, there were no mortality and no observable clinical signs of ferrocene-related toxicity in any of the animals. At the end of the exposures, male rats exposed to the highest level of ferrocene had decreased body-weight gains relative to the weight gained by filtered air-exposed control rats, while body-weight gains for all groups of both ferrocene- and filtered air-exposed female rats were similar. Male mice exposed to the highest level of ferrocene also had decreased body-weight gains, relative to controls, while female mice had relative decreases in body-weight gains at the three highest exposure levels. Male rats had a slight decrease in relative liver weight at the highest level of exposure, whereas no relative differences in organ weights were seen in female rats. Male mice had exposure-relative decreases in liver and spleen weights, and an increase in thymus weights, relative to controls. For female mice, relative decreases in organ weights were seen for brain, liver, and spleen. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Histopathological examination was done only on the nasal turbinates, lungs, liver, and spleen. The only exposure-related finding was histopathologic lesions in the nasal turbinates of both species. These lesions were primarily centered in the olfactory epithelium and were morphologically diagnosed as subacute, necrotizing inflammation. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. In vitro metabolism studies of ferrocene showed that nasal tissue, particularly the olfactory epithelium, had 10 times higher “ferrocene hydroxylating” activity than did liver tissue from the same animals. These results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by iron-catalyzed lipid peroxidalion of cellular membranes.

Antimony potassium tartrate (APT) is a complex salt that until recently was used worldwide as an antischistosomal drug. Treatment was efficacious only if APT was administered intravenously to humans at a near lethal total dose of 36 mg/kg. Because unconfirmed epidemiologic studies suggested there might be an association between APT treatment and bladder cancer, we initiated prechronic toxicity studies with the drug to select a route of administration and doses in the event that chronic studies of APT were needed. The toxicity and concentration of tissue antimony levels were compared in 14-d studies with F344 rats and B6C3F1 mice administered APT in the drinking water or by ip injection to determine the most appropriate route for longer term studies. Drinking water doses estimated by water consumption were 0, 16, 28, 59, 94 and 168 mg/kg in rats and 0, 59, 98, 174, 273, and 407 mg/kg in mice. APT was poorly absorbed and relatively nontoxic orally, whereas ip administration of the drug caused mortality, body weight decrements, and lesions in the liver and kidney at doses about one order of magnitude below those in drinking water. Because of these data and the dose-related accumulation of antimony in the target organs, an ip dose regimen was selected for subsequent studies. Both sexes of F344 rats and B6C3F1 mice were given 0, 1.5, 3, 6, 12, and 24 mg/kg doses of APT every other day for 90 d by ip injection. There were no clinical signs of toxicity nor gross or microscopic lesions in mice that could be attributed to toxicity of APT, although elevated concentrations of antimony were detected in the liver and spleen of mice. Rats were more sensitive than mice to the toxic effects of APT, exhibiting dose-related mortality, body weight decrements, and hepatotoxicity. The concentrations of antimony measured in liver, blood, kidney, spleen, and heart of rats were proportional to dose, but there were no biochemical changes indicative of toxicity except in the liver. Hepatocellular degeneration and necrosis occurred in association with dose-related elevations in activities of the liver-specific serum enzymes sorbitol dehydrogenase and alanine aminotransferase. By alternating the site of abdominal injection and the days of treatment, mesenteric inflammation at the site of administration was minimized in the rats and mice, indicating that the ip route would be suitable for chronic studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Groups of 50 F344/N rats of each sex and 50 B6C3F1 mice of each sex were gavaged with corn oil or a mixture of toluene diisocyanate (TDI) in corn oil for 5 days per week for 105 or 106 weeks. Female rats and mice were given doses of 60 or 120 mg/kg body weight, while male rats received 30 or 60 mg/kg, and male mice received 120 or 240 mg/kg. The TDI reacted with the moisture in the corn oil vehicle resulting in doses that were 10% to 23% below the target dose concentrations. The chemical product used was commercial grade TDI, which was an 80%-20% mixture of the 2,4- and 2,6-isomers. Chemical disposition and metabolism studies were conducted with each of the radiolabelled TDI isomers in male rats. Absorption of both of the TDI isomers occurred, with the highest concentrations found in the stomach, cecum, large intestine, and bladder. Excretion occurred via the feces and urine. The major metabolic products from the metabolism of 2,4-TDI were shown to be identical with those from the metabolism of the carcinogen, 2,4-diaminotoluene, whereas the metabolism of the 2,6-TDI isomer yielded one major product, identified as 2,6-bis(acetylamino)toluene. Greater than 10% depression in body weight gain occurred in all dosed groups of rats throughout most of the study. The major non-neoplastic lesions that were observed in both sexes of the TDI-exposed rats were dose-related increases in acute broncho-pneumonia, characterized as chemical pneumonitis, with incidences as high as 50%. In mice mean body weight gain was depressed in dosed male and in high dose females. The principle non-neoplastic lesion in mice that was attributed to chemical treatment was cytomegaly of the kidney tubular epithelium in males. Survival in all groups of dosed rats was significantly lower than in controls. A dose-dependent pattern of mortality did not commence until 70 weeks of exposure, demonstrating that toluene diisocyanate elicited a cumulative toxic response. There was also significantly lower survival in high dose male, but not female mice, by comparison to controls. Despite the reduction of power and sensitivity in the rat studies caused by early mortality, statistically significant increases in tumor incidences were observed in many different target organs. TDI was carcinogenic in F344/N rats, causing subcutaneous fibromas and fibrosarcomas in males and females, pancreatic acinar cell adenomas in males, and pancreatic islet cell adenomas, neoplastic nodules of the liver, and mammary gland tumors in females.(ABSTRACT TRUNCATED AT 400 WORDS)

The efficacy of a leukemia cell transplant model to measure potential chemotherapeutic activity was tested with five different chemicals that had previously been evaluated in 2-year studies. Leukemic spleen cells from Fischer rats were injected subcutaneously into syngeneic recipients and the effects of chemical treatment on tumor progression were evaluated at 70 days post-transplant. The data from the short-term assay were in all cases correlated with the trends reported for mononuclear cell leukemia in 2-year studies, where two chemicals were reported to decrease the incidence and three chemicals were reported to increase the incidence of leukemia. Short-term treatment with the two chemicals which caused negative trends for leukemia (2-ethoxyethanol or ethylene glycol monoethyl ether; 4-hexylresorcinol) delayed and/or reduced tumor growth in the transplant model in a dose-related fashion, as exhibited by reduction or elimination of splenomegaly and leukoblastosis, and a reversal in the depression of red blood cell indices or platelet counts. By contrast, the rate of tumor progression was increased in the short-term assay of the three chemicals which previously caused increased trends for leukemia in 2-year studies (pyridine; 2,4,6-trichlorophenol, dichlorvos). The severity of the mononuclear cell leukemia in the transplant recipients, as measured by histopathological examination of spleen and liver, was correlated with the changes in tumor growth rates. The in vivo leukemia transplant model is a short-term assay that could be used to screen a variety of potential chemotherapeutic agents, or to study structure-activity relationships within one class of chemicals.

Female B6C3F1 mice were exposed to graded doses of nickel sulfate to determine a threshold response for myelotoxicity and immunotoxicity, and to identify which of the populations of lymphoreticular cells were most sensitive to the toxic effects of nickel. Animals were given free access to the chemical in the drinking water at 0, 1, 5, or 10 g/l for 180 d. Water consumption, blood and tissue nickel concentrations, body and organ weights, histopathology, immune responses, bone marrow cellularity and proliferation, and cellular enzyme activities were evaluated. There was no mortality. Mice in the 5-g/l and 10-g/l dose groups drank less water than controls; the responses measured in the 10-g/l group may have been due to a combination of dehydration and chemical toxicity. Decreases in body and organ weights were confined to mice in the 10-g/l dose group, except for the dose-related reductions in thymus weights. Blood nickel was measured at 4, 8, 16, and 23 wk of exposure. The mean blood nickel values showed increases between 4 and 8 wk that were proportional to time and dose; thereafter there was no substantial increase in blood nickel in any of the dose groups, except for an increase in the mean blood concentration in the 10-g/l group at 23 wk. The kidney was the major organ of nickel accumulation. The primary toxic effects of nickel sulfate were expressed in the myeloid system. There were dose-related decreases in bone marrow cellularity, and in granulocyte-macrophage and pluripotent stem-cell proliferative responses. In unfractionated bone marrow cells glucose-6-phosphate dehydrogenase enzyme activity from the hexose monophosphate shunt was more sensitive to nickel sulfate than were representative glycolytic or Krebs cycle enzymes, with 25-35% maximum inhibition at 5 g/l and 10 g/l. Aliquots of bone marrow cells were separated into enriched bands of lymphocytes, granulocyte-macrophages, and erythrocytes; enzyme inhibition that occurred in unfractionated bone marrow cell aliquots was only expressed after cell separation in the enriched granulocyte-macrophage cell population, suggesting that these committed stem cells were a primary target of nickel sulfate toxicity. There was one example of systemic immunotoxicity, reduction in the lymphoproliferative response to lipopolysaccharide, and it was regarded as secondary to the primary effect of nickel sulfate on the myeloid system, since this was the only significant change among a panel of seven immune parameters that were evaluated.

Female B6C3F1 mice plus male and female Fischer 344/N rats were gavaged with allyl isovalerate (AIV) in corn oil at 0, 31, 62, or 125 (mice) and 0, 31, 62, 125, or 250 (rats) mg/kg body weight for five daily exposures per week for a 2-week period. Hematologic, immunologic, and histopathologic studies were performed 48 to 72 hr following the final treatment. AIV exposure had no effect on hematology or bone marrow cellularity in mice or rats. AIV exposure at 250 mg/kg was toxic to rats causing reduced weight gain and hepatotoxicity. In vivo and in vitro studies revealed that pluripotent hematopoietic stem cells (CFU-S) and granulocyte-macrophage progenitors (CFU-GM) in the bone marrow were decreased in the treated mice. Hematopoietic suppression was correlated with the reduction in the hexose monophosphate shunt metabolism of bone marrow cells but the Embden-Meyerhof pathway and tricarboxylic acid pathway enzymes did not appear to be affected. Examination of host resistance following Plasmodium and Listeria challenge did not demonstrate significant differences between treated and control mice, nor were there other effects on the immune system. This suggests that the myelotoxic effects were minimal and of a degree that would not alter host resistance.

Cellular glucose-metabolizing enzymes and acetylcholinesterase (AChE) have been utilized as biochemical markers of mononuclear cell (MNC) leukemia maintained by serial cell transplantation in F344 rats. We have evaluated the sensitivity and reproducibility of these tumor markers in comparison to other diagnostic criteria of leukemia. Weanling rats were injected with 2 X 10(7) leukemic spleen MNC and sampled at 6, 35, 63, and 83 days. At 6 days, the glycolytic enzyme activities from spleen that decreased were believed to be residual activity from injected leukemic MNC. Glycolytic enzyme activities in spleen MNC were normal at 35 days and no changes in blood MNC enzyme activity occurred at 6 days or 35 days. At 63 days, prior to clinical evidence of leukemia, glucose-metabolizing enzymes from spleen MNC changed, and there were decreases in AChE from both blood and spleen MNC that progressively decreased at 83 days, when there was depressed body weight, splenomegaly, elevated WBC, depressed RBC, hypoglycemia, hyperbilirubinemia, and elevated serum enzyme levels. Separation of leukemic MNC from blood and spleen enhances sensitivity of cellular enzyme responses and provides a reproducible model to study biochemical markers correlated with severity of leukemia.

Compounds with estrogenic activity cause initial toxic responses in the bone marrow characterized by hypocellularity and stem cell myelotoxicity. To elucidate the biochemical nature of these toxic responses, bone marrow cells were collected from mice treated with pharmacological doses of estrogenic chemicals, separated into enriched cell populations, and the enzymatic responses of the individual cell types characterized. Female B6C3F1 mice were injected s.c. with five daily doses of 0.07-5.6 mu moles diethylstilbestrol (DES) or 17-beta estradiol. At 4 days posttreatment body and organ weights were recorded and bone marrow was collected for enumeration and assay of stem cell proliferative responses and enzyme analyses. Treatment with higher dose levels of either estrogenic chemical caused equivalent thymic atrophy, but DES resulted in greater liver and spleen hypertrophy than estradiol. Hexose monophosphate shunt dehydrogenase enzymes in unfractionated bone marrow cells were more sensitive to inhibition by lower estrogen doses than representative enzymes from glycolysis of the Kreb's Cycle, and on an equimolar basis were inhibited to a greater extent by DES than by estradiol. Enzyme analyses after density gradient cell separation indicated that 70-80% of the hexose monophosphate shunt enzyme activity in bone marrow from untreated mice occurred in the enriched band of cells containing predominantly granulocyte-macrophages. The majority of the enzyme inhibition induced by DES treatment could also be ascribed to this cellular population. Furthermore, it was shown that DES had a greater inhibitory effect on the proliferative capacity of the committed stem cells than on the multipotential stem cell population, and the main response was again expressed in the enriched band of cells containing predominantly granulocyte-macrophage precursors. Preliminary endocrine ablation experiments indicated estrogen inhibition of hexose monophosphate shunt enzyme activity was independent of the adrenal and the ovary, but was mediated through the thymus at lower estrogen concentrations.