Differential protection by rat UDP-glucuronosyltransferase 1A7 against Benzo[a]pyrene-3,6-quinone- versus Benzo[a]pyrene-induced cytotoxic effects in human lymphoblastoid cells.

UDP-glucuronosyltransferase 1A7 (UGT1A7) is a polyaromatic hydrocarbon (PAH)-inducible UGT with activity toward various benzo[a]pyrene (B[a]P) metabolites. To investigate the influence of rat UGT1A7 on B[a]P-induced cytotoxicity, human lymphoblastoid L3 cells were transfected with pMF6 (control expression vector), p167Dtk2 (microsomal epoxide hydrolase expression vector), or p167Dtk2-1A7 (epoxide hydrolase/UGT1A7 coexpression vector), and the cell populations were compared for sensitivity to B[a]P-induced effects. B[a]P inhibited cell proliferation and decreased relative cell survival of p167Dtk2 and p167Dtk2-1A7 cells to a similar extent. Metabolism studies using [(3)H]B[a]P revealed increased formation of glucuronide conjugates of B[a]P-4,5-diol, 3-OH-, or 9-OH-B[a]P and an unidentified metabolite by p167Dtk2-1A7 cells, but the presence of unconjugated metabolites suggested that glucuronidation capacity may be limited. No differences between p167Dtk2 and p167Dtk2-1A7 L3 cells were observed in the growth inhibitory effects of 3-OH-B[a]P or B[a]P-7,8-diol, but p167Dtk2-1A7-expressing cells were found to be less sensitive to B[a]P-3,6-quinone-induced effects on cell proliferation and relative cell survival. The effect was also observed in AHH-1 lymphoblastoid cells expressing UGT1A7 without epoxide hydrolase. The UGT1A7-expressing AHH-1 cells were also less sensitive to growth inhibition by B[a]P-1,6-quinone and B[a]P-6,12-quinone. Flow cytometric analysis of vehicle and B[a]P-3, 6-quinone-exposed cell populations showed an association between UGT1A7 expression and resistance to B[a]P-3,6-quinone-induced apoptosis and loss of cell viability. These data suggest that UGT1A7 may be preferentially active toward B[a]P-quinones and that UGT1A7 may represent the PAH-inducible UGT activity previously implicated in protection against toxic redox cycling by B[a]P-3,6-quinone.

A community outbreak of Legionnaires' disease linked to hospital cooling towers: an epidemiological method to calculate dose of exposure.

BACKGROUND: From July to September 1994, 29 cases of community-acquired Legionnaires' disease (LD) were reported in Delaware. The authors conducted an investigation to a) identify the source of the outbreak and risk factors for developing Legionella pneumophila serogroup 1 (Lp-1) pneumonia and b) evaluate the risk associated with the components of cumulative exposure to the source (i.e. distance from the source, frequency of exposure, and duration of exposure). METHODS: A case-control study matched 21 patients to three controls per case by known risk factors for acquiring LD. Controls were selected from patients who attended the same clinic as the respective case-patients. Water samples taken at the hospital, from eight nearby cooling towers, and from four of the patient's homes were cultured for Legionella. Isolates were subtyped using monoclonal antibody (Mab) analysis and arbitrarily primed polymerase chain reaction (AP-PCR). RESULTS: Eleven (52%) of 21 case-patients worked at or visited the hospital compared with 17 (27%) of 63 controls (OR 5.0, 95% CI : 1.1-29). For those who lived, worked, or visited within 4 square miles of the hospital, the risk of illness decreased by 20% for each 0.10 mile from the hospital; it increased by 80% for each visit to the hospital; and it increased by 8% for each hour spent within 0.125 miles of the hospital. Lp-1 was isolated from three patients and both hospital cooling towers. Based on laboratory results no other samples contained Lp-1. The clinical and main-tower isolates all demonstrated Mab pattern 1,2,5,6. AP-PCR matched the main-tower samples with those from two case-patients. CONCLUSION: The results of our investigation suggested that the hospital cooling towers were the source of a community outbreak of LD. Increasing proximity to and frequency of exposure to the towers increased the risk of LD. New guidelines for cooling tower maintenance are needed. Knowing the location of cooling towers could facilitate maintenance inspections and outbreak investigations.

Immunological evaluation of the mycotoxin patulin in female B6C3F1 mice.

Patulin is a mycotoxin produced by many fungal species of the genera Penicillium, Aspergillus and Bryssochamys. Previous literature reports have suggested that patulin is toxic to the immune system. The studies presented were conducted to provide a comprehensive assessment of the effects of patulin on the immune system. Unlike previous reports, the doses of patulin used (0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg/kg) were based on predicted human exposure levels. Female B6C3F1 mice were exposed orally to patulin for 28 days. Effects were not observed on final body weight or body weight gain. Relative weight of the liver, spleen, thymus, kidneys with adrenals, and lungs was not affected. Peripheral blood leucocyte and lymphocyte counts were decreased by approximately 30% in the two highest dose groups. The leucocyte differential was not altered. Total spleen cell, total T-cell (CD3+), helper T-cell (CD4+CD8-), B-cell (surface immunoglobulin+) and monocyte (MAC-3+) counts were not changed. Cytotoxic T-cell (CD8+CD4-) counts were increased 50% only by the highest dose. Natural killer cell (NK1.1+CD3-) and monocyte (MAC-1+) counts were increased 30% and 24%, respectively, only in the 0.08 mg/kg group. Humoral immune function as assessed by antibody-forming cell response and serum IgM titre to sheep erythrocytes, and cell-mediated immune function evaluated utilizing natural killer cell activity and the mixed lymphocyte reaction were not altered. Oral exposure to patulin for 28 days did not alter the ability of female B6C3F1 mice to mount either a cell-mediated or humoral immune response.

The effect of aflatoxin B1 exposure on serotonin metabolism: response to a tryptophan load.

Semi-chronic exposure of ICR male Mice to Aflatoxin B1 (AFB1) in non-toxic doses decreased brain serotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and catecholamines without altering tryptophan (TRP) or tyrosine (TYR) levels. A TRP load (300 mg/kg, i.p. x 2 hours) slightly increased brain TRP levels while causing a slight decrease in 5-HT and 5-HIAA in control animals. A TRP load in AFB1 treated mice increased brain 5-HT and 5-HIAA. The TRP load caused a further reduction in brain catecholamines without altering TYR levels. Exposure to AFB significantly increased lung TRP levels without altering 5-HT or 5HIAA levels. TRP loading increased lung TRP concentrations in control mice. However, in AFB1 treated mice the increase was not significantly elevated above the level caused by AFB1 treatment alone. Lung 5-HT or 5-HIAA levels in control or AFB1 treated mice are not significantly altered by TRP loading. These experiments demonstrate that AFB1 alters brain and lung TRP metabolism.

Lead exposure in outdoor firearm instructors.

This study was conducted to determine lead exposure of firearm instructors at an outdoor firing range, while cadets were firing nonjacketed and jacketed lead ammunitions. The breathing zone air for lead exceeded the Occupational Safety and Health Administration standard of 50 micrograms/m3 for two instructors during firing exercises using nonjacketed bullets. The use of totally copper-jacketed bullets reduced the breathing zone lead levels by 92 percent for instructor #1 and by 96 percent for instructor #2; subsequent blood lead levels showed a significant decline in both instructors.

Reducing exposures to airborne lead in a covered, outdoor firing range by using totally copper-jacketed bullets.

A study was conducted to evaluate the feasibility of reducing or eliminating airborne lead concentrations at a covered, outdoor firing range by using totally copper-jacketed ammunition. Used in the study were 38-caliber-special police revolvers, and firings were conducted with 38-caliber, totally copper-jacketed bullets. The mean lead levels in general area air samples and personal breathing zone samples were 9.53 micrograms/m3 and 5.88 micrograms/m3, respectively, calculated as an 8-hr, time-weighted average (TWA). The mean copper levels in general area samples and personal breathing zone samples were 0.8 micrograms/m3 and 1.43 micrograms/m3, respectively. These concentrations in general area air samples and personal breathing zone samples were well below the current Occupational Safety and Health Administration (OSHA) permissible exposure limit (PEL) for occupational exposure to inorganic lead (50 micrograms/m3) and for copper (100 micrograms/m3). No significant difference was found between blood lead levels before and after firing totally copper-jacketed bullets. Based on comparison with results from a comparable study using nonjacketed lead ammunition, it was concluded that the use of totally copper-jacketed bullets significantly reduced airborne lead levels by a factor of 21 in the personal breathing zone samples and by a factor of 7.5 in the general area air samples.

Acute and chronic effects of the trichothecene mycotoxin T-2 on rat brain regional concentrations of serotonin, tryptophan, and tyrosine.

Female rats were given 1 acute dose or chronic doses (once every 48 hr for 28 days) of T-2 toxin (10 micrograms/kg ip) or vehicle. At necropsy, each brain was subdivided into cerebellum, cerebral cortex (including telencephalon and diencephalon), and brainstem (including mesencephalon, metencephalon, and myelencephalon). Acute systemic T-2 toxin administration increased cerebellar and brainstem tryptophan while serotonin, a tryptophan metabolite, was decreased correspondingly in these same brain regions. Chronic T-2 administration increased cerebellar tyrosine and serotonin concentrations, while cortical tryptophan concentrations were also increased. These results indicate that both acute and chronic administration of T-2 toxin cause differential changes in regional distribution levels of tyrosine, tryptophan, and serotonin.

Differential changes in rat brain tryptophan, serotonin and tyrosine levels following acute aflatoxin B1 treatment.

Female Sprague-Dawley rats were treated acutely (12-h) with aflatoxin B1 (100 micrograms/kg i.p.) or vehicle (10% acetone in 0.9% NaCl) and regional brain levels of tryptophan, serotonin and tyrosine were assayed. Brainstem but not cerebellar or cortical tyrosine levels were decreased in aflatoxin B1-treated rats. Brain tryptophan was increased in all 3 brain regions by acute aflatoxin B1 treatment, while serotonin levels were unaltered in the cerebellum and cortex and decreased in the brainstem. These experiments indicate that acute aflatoxin B1 treatment differentially alters brain amino acids and serotonin and that changes in brain tryptophan, the serotonin precursor, do not parallel changes in brain serotonin.

Further evidence supporting the concurrent influence of aflatoxin and manganese.

Trace elements, including manganese may afford protection from deleterious effects of aflatoxin. Young male Fischer rats received ip injections of aflatoxin B1 (AFB1) in dimethyl sulfoxide (DMSO), 1 mg/kg, 2 mg/kg or 4 mg/kg. Control groups received DMSO ip or no injection. All animals were intubated with 3 microCi of [54Mn]-MnCl2 12 hr post-injection. Sacrifice occurred 72 hr after gavage of the radiolabel. All tested levels of AFB1 affected the loss of total body radioactivity. This response was observed within 12 hr when toxin-treated groups excreted almost 4 times more counts than controls. From 12-36 hr following radiolabel administration, AFB1 appeared to enhance excretion; by 72 hr, toxin-treated animals (especially those receiving higher doses) appeared to conserve the metal. Aflatoxicosis manifested itself through reduced body weight gain. The data provide support evidence that Mn and AFB1 biointeract.

Reduced aflatoxicosis in livers of hamsters fed a manganese sulfate supplement.

Male, weanling Syrian hamsters (Mesocricetus auratus) were given (for 10 weeks) diets supplemented with manganese sulfate, aflatoxin, or a combination of both. All animals were killed and a histopathologic evaluation was performed on each liver to assess the influence of a manganese-supplemented diet on aflatoxicosis. Serum cholesterol and liver glycogen levels were also analyzed to further study the interaction of manganese and aflatoxin. Characteristic aflatoxin-induced precancerous histopathologic changes were observed in animals receiving the toxin. These changes included bile duct cell hyperplasia, enlarged nuclei, nuclear inclusions, and megala-hepatocytes. The dietary manganese addition to aflatoxin animals caused a slight reduction in the bile duct cell hyperplasia and significantly reduced the enlarged nuclei and nuclear inclusions. The latter indicates that the manganese may be influencing membrane chemistry. Animals receiving aflatoxin alone showed significantly increased serum cholesterol and liver glycogen. The cholesterol levels were significantly increased over the aflatoxin-induced levels when manganese was given in combination with the aflatoxin. The manganese lowered the increased liver glycogen levels caused by the aflatoxin. Dietary manganese shows some potential in suppressing several, but not all, of the aspects of developing aflatoxicosis in the hamster. The specific mode and site of action of manganese requires additional study.

Aflatoxin-induced alterations in Glycine max, cv. 'Essex' root cell membrane protein content.

Aflatoxins (AFTs) are hepatocarcinogens, mutagens, teratogens and toxins. Isolates of AFTs-producing strains of Aspergillus flavus can grow upon both autoclaved soybeans and to a lesser extent field-grown beans. Besides inhibiting the elongation of excised, in vitro cultured soybean roots, the AFTs can impair the roots' ability to remove [14C]-leucine from a culture medium and to incorporate the amino acid into acid-insoluble, cytoplasmic protein. In this connection, exogenous AFTs once taken-up and compartmentalized by the excised roots can reduce the acid-insoluble protein content of what appears to be a non-enriched plasmalemma fraction. Here, we report a combined biochemical and microscopical attempt to determine whether the protein reduction occurs throughout the excised, in vitro-cultured root and whether the plasmalemma proteins which are affected by AFTs can be both solubilized and characterized. Histochemistry of Carnoy-fixed roots revealed a reduction in Ninhydrin-Schiff-stainability throughout the AFTs-treated root. Transmission electron microscopy of 80 000 X g pellets derived from homogenates of both non-treated (NT) and treated (T) roots provided further evidence to our previously reported marker enzyme analyses for the occurrence of the plasmalemma in the 80 000 X g pellet. Treatment of the latter with the Laemmli SDS procedure released greater than 85% of the protein associated with the pellets obtained from homogenates of either NT or T roots. Gel permeation chromatography on Biogel-100 of released proteins from 80 000 X g pellets of both NT and T roots yielded both void volume and retarded peaks. Both the amplitude and the quantities of protein recovered within the void volume peak were less within the void volume of the t than the NT root. Polyacrylamide tube gel electrophoresis of G-100 void volume peaks of chromatographed, SDS-released proteins from the pellets revealed quantitative but not qualitative differences in Coomassie Blue-gel staining patterns between T and NT roots. These results suggest that the SDS methods which we employed could solubilize the 80 000 X g pellet proteins but that combined gel permeation chromatography and tube gel electrophoresis were not sensitive enough to reveal whether AFTs could alter the types of proteins associated with the 80 000 X g pellet (purported plasmalemma).

Disturbances in tryptophan metabolism in rats following chronic dietary aflatoxin treatment.

Male Holtzman rats were fed 7.8 ppm mixed aflatoxins (AFT) for a 21-week period. At sacrifice there was extensive bile ductule cell proliferation and numerous precancerous changes evident in AFT-treated animals. The in vitro activity of hepatic holo-enzyme tryptophan 2, 3-dioxygenase was reduced in AFT-treated animals which correlated with a reduced urinary excretion of both kynurenine and kynurenic acid suggesting that this reduced tryptophan 2, 3-dioxygenase observed in vitro may be relevant to the in vivo response. The levels of serotonin and 5-hydroxyindole-3-acetic acid were found to be reduced in the pineal, pituitary, various brain regions and in duodenum of AFT-treated rats. It is proposed that the decreased levels of serotonin and 5-hydroxyindole-3-acetic acid in all tissues may be due to either a decreased free tryptophan pool or alterations in 5-hydroxyindole biosynthesis.

Evaluation of the toxic effects of aflatoxin B1 with a taste aversion paradigm in rats.

A taste aversion test was used to evaluate the toxic effects of aflatoxin (AF) B1 which is considered to be one of the most potent hepatocarcinogens known. Sixty male Fisher rats were randomly assigned to six treatment (injection) groups: Water (H2O); Dimethyl Sulphoxide (DMSO); Lithium Chloride (LiCl); 0.125 mg/kg AFB1 (AF-Low); 0.250 mg/kg AFB1 (AF-Medium) and 0.500 mg/kg AFB1 (AF-High). After adaption to a water-deprivation schedule which permitted 10-min access to water each day, the rats were given 10-min access to 0.1% saccharin solution which was followed by intraperitoneal injections of either H2O, DMSO, LiCl, or AF of varying doses according to group assignment. After a five-day recovery period, the rats were initially tested for a conditional taste aversion to the saccharin solution with a two-bottle test during the 10-min access period. Then, a series of two-bottle taste aversion tests were conducted in which H2O and saccharin solutions were available on an ad lib basis. The LiCl, AF-Low, AF-Medium, and AF-High groups developed strong aversions to the saccharin solution; whereas, the H2O and DMSO control groups did not. In addition, there was a dose-response relationship between the doses of aflatoxin and the level of aversion with the AF-High group demonstrating the most marked aversion throughout all saccharin tests. Retention tests showed that the taste aversion was still evident 30 days after the initial pairing of saccharin with AF.

Activities of tryptophan-metabolizing enzymes in liver and brain of rats treated with aflatoxins.

Male Sprague-Dawley rats were treated ip twice daily for 10 days with mixed aflatoxins (10 micrograms/kg body weight) and then the activities of liver tryptophan 2,3-dioxygenase and brainstem tryptophan 5-hydroxylase were determined in vitro. Total tryptophan 2,3-dioxygenase activity was reduced by aflatoxin treatment while holoenzyme activity was not. Induction of tryptophan 2,3-dioxygenase activity by L-tryptophan was not altered by the treatment though induction by hydrocortisone was blocked. It is suggested that aflatoxin may alter the tryptophan 2,3-dioxygenase-haem bond in vivo rather than affect enzyme or cofactor synthesis. Kinetics studies performed in vitro on brain tryptophan 5-hydroxylase showed that aflatoxin treatment in vivo increased the Km of the hydroxylase when L-tryptophan and synthetic 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin were used as the substrate and cofactor, respectively. However, aflatoxin treatment did not alter the Vmax of tryptophan-5-hydroxylase.

Aflatoxin and dimethyl sulfoxide influence on radiomanganese distribution and retention in neonate mice.

The LD50 (7 d) for aflatoxin B1 (AFB1) in CD-1 neonate mice (3.1 g; 5 d of age) was determined to be 13.3 mg/kg. The vehicle was dimethyl sulfoxide (DMSO), given intraperitoneally, at 0.01 ml/animal (7 mg/kg). The solvent was nontoxic and caused no significant change in body weight in animals during an 11-d experimental period (17 d of age). Aflatoxin B1 at 5.0 mg/kg and above caused reduced body weight gain. DMSO animals had a mean loss of more than 17% of the radiolabel over a 9-d period. Aflatoxin treatments reversed the DMSO loss of 54Mn in a concentration-related fashion, and generally, AFB1 caused a conservation of the radioisotope. The radiolabel was redistributed into the following organs/tissues: liver greater than brain greater than bone greater than muscle = lungs greater than blood. Aflatoxin-treated animals showed a twofold increase of radiolabel in the liver as compared to controls. The DMSO itself failed to influence 54Mn influx into the liver. In general, control neonate mice, by 17 d of age, were retaining and redistributing the 54MnCl2 and had not reached the time for sudden emergence of excretion common in rodents. DMSO was found not to be the most satisfactory solvent to use in the administration of aflatoxins, especially when manganese metabolism is being studied. Generally, both DMSO and AFB1 influenced radiomanganese distribution, DMSO having a substantial influence.

Histochemical analysis of liver cells from short term, aflatoxin-dosed and nondosed Coturnix coturnix japonica. 1. Aflatoxin-sensitive quail.

Although aflatoxicosis in Coturnix coturnix japonica has been described, the histochemical localization of liver chemicals and the occurrence of ingested aflatoxins within blood, feces, and liver have not been described. Six to 8-week-old quail, which were intubated with a carrier with or without .3 mg mixed aflatoxins (AFB1, AFB2, AFG1, AFG2)/kg body weight were sacrificed within .25 to 5 days of intubation. Deparaffinized sections of livers were stained for lipids, nucleic acids, polysaccharides, and proteins. Other livers and excrement were homogenized and filtered homogenates as well as blood partitioned against chloroform. The aqueous phase was treated with pepsin and then partitioned, but the organic phase was analyzed directly. Organic phases of .25 to 5 day blood, feces, and liver lacked aflatoxins. Pepsin digesta of blood from males and females dosed 6 hr appeared to contain aflatoxicol (disappeared by 24 hr) and an unknown fluorescent compound, respectively. Whereas an unidentified fluorescent compound was observed within excrement of males dosed 6 hr, female excrement contained a fluorescent compound with an AFB1 Rf (disappeared by 24 hr). Although the liver of males dosed 6 hr had three fluorescent compounds (Rfs for AFB1 and AFB2a), only one was seen within dosed females. Ultra violet absorption spectra of presumed AFB2a and aflatoxicol failed to yield their reported absorption maxima. Livers from dosed quail exhibited bile duct proliferation, cellular necrosis, vacuolization, congestion, fatty changes and mild hepatitis. Sinusoidal membranes were thickened and contained abundant periodic acid-Schiff's (PAS)-positive substances. Although livers of nondosed quail abounded with regularly shaped and uniformly distributed, Sudan IV-positive droplets, livers of dosed quail accommodated few, irregularly-shaped and positioned droplets. Hepatocyte nuclei and nucleoli of dosed quail displayed marked affinities for the Feulgen reagent and toluidine blue O, respectively. Lobules of dosed quail possessed concentrations of cells in which their entire cytoplasm was PAS positive. Treatment of sections with alpha-amylase reduced staining suggesting the presence of glycogen. Ninhydrin-positive substances were distributed throughout the liver in both DQ and non DQ with no apparent difference in intensities between the two livers. Generally the DQ showed mild hepatitis due to aflatoxicosis and the toxin altered liver histochemistry for the major classes of cellular chemicals.