June K. Dunnick

Herpes Simplex Encephalitis: Vidarabine Therapy and Diagnostic Problems

To learn more about the treatment of herpes simplex encephalitis with vidarabine, we conducted an uncontrolled study of 132 patients referred to 22 hospitals because of suspected disease. All had a brain biopsy and were started on vidarabine, but only 75 were diagnosed by isolation of virus from a brain-biopsy specimen. Cumulative mortality in the latter group was 39 per cent at one year. Other than therapy, levels of consciousness and age were the major variables that influenced outcome. Of 23 patients under 30 years of age who were lethargic at the initiation of therapy, two died and 16 returned to normal. Of 26 patients over 30 years of age who were lethargic at the outset, nine died and 10 returned to normal. Semicoma and coma were associated with worse outcomes, especially in older patients. Our data suggest that outcome is improved with treatment; they provide more support for the use of brain biopsy to diagnose the infection and indicate a need for better therapy.

Phenolphthalein Induces Thymic Lymphomas Accompanied by Loss of the p53 Wild Type Allele in Heterozygous p53-Deficient (±) Mice

Epidemiology studies have indicated that many human cancers are influenced by environmental factors. Genetically altered mouse model systems offer us the opportunity to study the interaction of chemicals with genetic predisposition to cancer. Using the heterozygous p53-deficient (±) mouse, an animal model carrying one wild type p53 gene and one p53 null allele, we studied the effects of phenolphthalein on tumor induction and p53 gene alterations. Earlier studies showed that phenolphthalein caused carcinogenic effects in Fisher 344 rats and B6C3F, mice after a 2-yr dosing period (Dunnick and Hailey, Cancer Res. 56: 4922-4926, 1996). The p53 (±) mice received phenolphthalein in the feed at concentrations of 200, 375, 750, 3,000, or 12,000 ppm (approximately 43, 84, 174, 689, or 2,375 mg/kg body weight/day or 129, 252, 522, 2,867, or 7,128 mg/m2 body surface area/day) for up to 6 mo. A target organ cancer site that accumulated p53 protein in the B6C3F, mouse (i.e., thymic lymphoma) was also a target site for cancer in the p53 (±) mouse. In the p53 (±) mouse, treatment-related atypical hyperplasia and malignant lymphoma of thymic origin were seen in the control and dosed groups at a combined incidence of 0, 5, 5, 25, 100, and 95%, respectively. Twenty-one of the thymic lymphomas were examined for p53 gene changes, and all showed loss of the p53 wild type allele. Chemical-induced ovarian tumors in the B6C3F, mouse showed no evidence for p53 protein accumulation and did not occur in the p53 (±) mouse. The p53-deficient (±) mouse model responded to phenolphthalein treatment with a carcinogenic response in the thymus after only 4 mo of dosing. This carcinogenic response took 2 yr to develop in the conventional B6C3F, mouse bioassay. The p53-deficient (±) mouse is an important model for identifying a carcinogenic response after short-term (<6 mo) exposures. Our studies show that exposure to phenolphthalein combined with a genetic predisposition to cancer can potentiate the carcinogenic process and cause p53 gene alterations, a gene alteration found in many human cancers.

Comparative inhalation toxicity of nickel subsulfide to F344/N rats and B6C3F1 mice exposed for 12 days.

Groups of F344/N rats and B6C3F1 mice were exposed to aerosols of nickel subsulfide (Ni3S2) 6 hr/day for 12 days not including weekends. Actual exposure concentrations were within 3% of target (target = 10.0, 5.0, 2.5, 1.2, 0.6, and 0.0 mg Ni3S2/m3). Nickel lung burdens of exposed rats and mice increased linearly with exposure concentration. Two male rats and all mice exposed to 10.0 mg Ni3S2/m3 died before the end of the exposures. Exposure to Ni3S2 had no effect on the natural killer cell activity of mouse spleen cells. Lesions in rats and mice related to inhalation of Ni3S2 were found in the nasal epithelium, lung, and bronchial lymph nodes. The most extensive lesions were found in the lung and included necrotizing pneumonia. Emphysema developed in rats exposed to 5.0 or 10.0 mg Ni3S2/m3, while fibrosis developed in mice exposed to 5.0 mg Ni3S2/m3. Degeneration of the respiratory epithelium and atrophy of the olfactory epithelium of the nose occurred in rats exposed to as low as 0.6 mg Ni3S2/m3 and mice exposed to 1.2 mg/m3. Results indicate that inhalation exposure of rats and mice to Ni3S2 aerosol concentrations near the current threshold limit value (TLV) for nickel compounds (1 mg/m3 for Ni metal and roasting fume and dust and 0.1 mg/m3 as Ni for soluble compounds) can produce lesions in the respiratory tract. Atrophy of lymphoid tissues (spleen, thymus, and bronchial lymph nodes) was found in animals of the highest exposure concentration. Degeneration of the testicular germinal epithelium was also observed in mice and rats that survived 5.0 or 10.0 mg/m3 exposure concentrations.

Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.

Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG‐p53® mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time‐weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, ripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN‐PCE and MN‐NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6‐monty treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose‐dependent increase in the frequency of MN‐PCE and MN‐NCE and in %PCE. Maximal induction of MN‐PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration‐dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not peexclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and (%PCE) among total erythrocytes, and the extent of 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN‐NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans. Environ. Mol. Mutagen. 31:113–124, 1998 Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Biochemical responses of rat and mouse lung to inhaled nickel compounds

Nickel subsulfide (Ni3S2), nickel sulfate (NiSO4), and nickel oxide (NiO) are encountered occupationally in the nickel refining and electroplating industries, with inhalation being a common route of exposure. The purposes of this study were to evaluate the biochemical responses of lungs of rats and mice exposed for 13 weeks to occupationally relevant aerosol concentrations of Ni3S2, NiSO4, and NiO, to correlate biochemical responses with histopathologic changes, and to rank the compounds by toxicity. Biochemical responses were measured in bronchoalveolar lavage fluid (BALF) recovered from lungs of exposed animals. Parameters evaluated in BALF were lactate dehydrogenase (LDH), beta-glucuronidase (BG), and total protein (TP). Total and differential cell counts were performed on cells recovered in BALF. All compounds produced an increase in LDH, BG, TP, and total nucleated cells, and an influx of neutrophils, indicating the presence of a cytotoxic and inflammatory response in the lungs of exposed rats and mice. Increases in BG were greater than increases in LDH and TP for both rats and mice. Chronic active inflammation, macrophage hyperplasia, and interstitial phagocytic cell infiltrates were observed histologically in rats and mice exposed to all compounds. Statistically significant increases in BG, TP, neutrophils, and macrophages correlated well with the degree of chronic active inflammation. Results indicated a toxicity ranking of NiSO4 greater than Ni3S2 greater than NiO, based on toxicities of the compounds at equivalent mg Ni/m3 exposure concentrations.

Comparative inhalation toxicity of nickel sulfate to F344/N rats and B6C3F1 mice exposed for twelve days.

Groups of F344/N rats and B6C3F1 mice were exposed to aerosols of nickel sulfate hexahydrate (NiSO4.6H2O) 6 hr/day for 12 days to determine the short-term inhalation toxicity of this compound. Target exposure concentrations were 60, 30, 15, 7, 3.5, and 0 mg NiSO4.6H2O/m3. Endpoints evaluated included clinical signs, mortality, quantities of Ni in selected tissues, effect on mouse resistance to tumor cells, and pathological changes in tissues of both rats and mice. All mice exposed to 7 mg NiSO4.6H2O/m3 or greater and 10 rats exposed to 15 mg NiSO4.6H2O/m3 or greater died before the termination of exposures. Quantities of Ni remaining in lungs of rats at the end of the exposure were independent of exposure concentration. Lung burdens of Ni in mice were approximately one-half that in lungs of rats. Exposure of female mice to 3.5 mg NiSO4.6H2O/m3 had no effect on resistance to tumor cells as determined by spleen natural killer cell activity. Histopathological changes were seen in tissues of rats and mice exposed to as low as 3.5 mg NiSO4.6H2O/m3. Lesions related to NiSO4.6H2O exposure occurred in lung, nose, and bronchial and mediastinal lymph nodes. Results indicated that exposure of rats and mice to amounts of NiSO4.6H2O aerosols resulting in Ni exposure concentrations only eight times greater than the current threshold limit value for soluble Ni (0.1 mg/m3) for as little as 12 days can cause significant lesions of the respiratory tract.

Cardiotoxicity of Ma Huang/Caffeine or Ephedrine/Caffeine in a Rodent Model System

Ma Huang (equivalent to 0, 12.5, 25, or 50 mg/kg ephedrine) or ephedrine (0, 6.25, 12.5, 25 mg/kg) were administered as one bolus oral dose to male F344 rats with and without caffeine. The herbal medicine Ma Huang (ephedra) in combination with caffeine caused rapid clinical signs of toxicity including salivation, hyperactivity, ataxia, and eventually lethargy, and failure to respond to stimuli. When this syndrome of clinical signs emerged, animals were moribund sacrificed, and a histological analysis for heart lesions performed. Cardiotoxicity included hemorrhage, necrosis, and degeneration in the ventricles or interventricular septum within 2–4 hours after treatment with Ma Huang (ephedra)/caffeine or ephedrine (the principal active component in Ma Huang)/caffeine. There was a steep dose response curve for cardiotoxicity with minimal toxicity seen at levels of Ma Huang (equivalent to 12.5 mg/kg ephedrine) with caffeine. However, cardiotoxic lesions occurred in 28% of animals with Ma Huang dosages equivalent to 25 mg/kg ephedrine with 15 or 30 mg/kg caffeine, and in 90% of animals at Ma Huang exposures equivalent to 50 mg/kg ephedrine with 15 or 30 mg/kg caffeine. Cardiotoxic lesions occurred in 47% of animals in the 25 mg/kg ephedrine groups with caffeine at 7.25, 15, or 30 mg/kg. There was no statistical difference in the occurrence of cardiotoxic lesions when 15 or 30 mg/kg caffeine was combined with Ma Huang equivalent to 25 or 50 mg/kg ephedrine; likewise there was no statistical difference in the occurrence of cardiotoxic lesions when 7.25, 15, or 30 mg/kg caffeine was combined with 25 mg/kg ephedrine. These results show that the cardiotoxic effects of the herbal medicine, Ma Huang, are similar to that of ephedrine, the principal active ingredient in the herbal medicine. The combination of Ma Huang or ephedrine with caffeine enhanced the cardiotoxicity over that with the herbal medicine or the active ingredient alone.

Experimental studies on the long-term effects of methylphenidate hydrochloride

Toxicology and carcinogenesis studies of methylphenidate hydrochloride, a drug used in the treatment of attention-deficient disorders, were performed in F344 rats and B6C3F1 mice. In these studies, methylphenidate hydrochloride was administered for 2 years at doses of 0, 100, 500 or 1000 ppm in the feed to rats and at doses of 0, 50, 250, 500 ppm to mice in groups that consisted of 50 animals/dose/sex/species. The average amount of methylphenidate consumed per day was estimated to be 4-47 mg/kg/day for rats and 5-67 mg/kg/day for mice. Survival was similar in dosed and control groups. An increase in benign tumors of the liver and increased liver weights were observed in male and female mice at the high dose. An increase in hepatoblastomas was also seen in high dose male mice. Methylphenidate was not mutagenic in the Salmonella assay system, and it is hypothesized that this tumorigenic effect might be due to nongenotoxic effects of the chemical such as an increase in cell proliferation. Increased incidences of neoplasms were not seen in rats. However, there was a notable decrease in mammary gland fibroadenomas in female rats and a marginal decrease in benign pheochromocytomas in male rats. Epidemiology studies of methylphenidate have found no evidence of a carcinogenic effect in humans and like our findings in rats, report a less than expected rate of cancers in patients taking methylphenidate.

Characterization of liver toxicity in F344/N rats and B6C3F1 mice after exposure to a flame retardant containing lower molecular weight polybrominated diphenyl ethers.

Lower molecular weight polybrominated diphenyl ethers (PBDEs), components of flame retardants, are found in the environment and in human and animal tissues. Toxicity studies were conducted in F344/N rats and B6C3F1 mice by administering a flame retardant containing these lower molecular weight PBDEs (BDE-47, BDE-99, BDE-100, and BDE153) by oral gavage 5 days/week for 13 weeks at doses of 0.01, 5, 50, 100 or 500mg/kg/day. Liver was the primary target organ in rats and mice. Treatment-related increases in liver weights, liver cytochrome P450 (1A1, 1A2, 2B) and UDPGT (rats only) levels, and liver lesions were seen in both rats and mice. Hepatocyte hypertrophy and vacuolization increased in incidence and severity with treatment, and occurred at levels of 50mg/kg and above in rats, and at 100mg/kg and above in mice. Liver Cyp 1A1, 1A2, and 2B levels were increased at exposure levels of 50mg/kg and above in rats and mice. In addition, treatment-related thyroid lesions occurred particularly in rats. The most sensitive parameter for PBDE toxicity was the increase in liver weights which occurred at 5mg/kg above in rats and 50mg/kg and above in mice. These results suggest that liver may be a target organ for carcinogenesis processes after long-term administration of PBDEs. A chronic PBDE study is currently being conducted by the National Toxicology Program.

Fate of Inhaled Nickel Oxide and Nickel Subsulfide in F344/N Rats

AbstractThe fates of inhaled nickel oxide (NiO, green oxide calcined at 1200°C) and nickel subsulfide (Ni3S2), two occupationally relevant nickel compounds, have been studied in male F344/N rats. Groups of rats underwent pernasal exposure to 9.9 mg NiO/m3 or to 5.7 mg Ni3S2/m3 for 70 and 120 min, respectively. The activity median aerodynamic diameters (geometric standard deviation) of the NiO and Ni3S2 aerosols were 1.3 μm (2.0) and 1.3 μm (1.5), respectively. End points evaluated included total and regional respiratory tract deposition of the aerosols, lung clearance of deposited material, distribution of solubilized material to extrarespiratory tract tissue, and pathways of Ni excretion from the body. The fractions of the inhaled NiO and Ni3S2 aerosols that deposited in the respiratory tract were 0.11 and 0.13, respectively. The fractions of the inhaled aerosol that deposited in the lungs were 0.05 for both aerosols. Inhaled NiO cleared slowly from the lungs, with a half-life of approximately 120 days. ...