Heat inactivation of Escherichia coli O157:H7 in apple cider containing malic acid, sodium benzoate, and potassium sorbate.

The effect of pH modification and preservative addition in apple cider on the heat resistance of Escherichia coli O157:H7 was investigated. E. coli O157:H7 and various amounts of potassium sorbate (0 to 0.2%), sodium benzoate (0 to 0.2%), and malic acid (0 to 1%) were added to apple cider. Thermal inactivation experiments were performed at 47, 50, and 53 degrees C, and D- and z-values were calculated. In apple cider without additives, the D-value at 50 degrees C (D50) was about 65 min, but addition of preservatives and malic acid significantly (P < 0.01) decreased D-values. D50-values decreased to 13.9 min in cider with 0.5% malic acid, 13.2 min with 0.1% sorbate, and 7.0 min with 0.1% benzoate added. Addition of both sorbate and malic acid had similar effects as either one alone, thus additive effects were not present. However, addition of both 0.2% benzoate and 1% malic acid did show additive effects, lowering D50 to 0.3 min. Addition of all three components (0.2% sorbate, 0.2% benzoate, and 1% malic acid) resulted in a D50 = 18 s. The z-value of cider without additives was about 6 degrees C, whereas z-values of cider containing malic acid, benzoate, and/or sorbate ranged from about 6 degrees C to 26 degrees C. This increase may result in a longer 5-log reduction time at higher temperatures (i.e., 70 degrees C) in cider with benzoate as compared to cider without additives.

Cross-fostering study of methyl mercury retention, demethylation and excretion in the neonatal hamster.

The cross-fostering technique was used in order to compare methyl mercury (MeHg) metabolism in hamsters following prenatal (in utero) and neonatal (lactational) exposure. Pregnant Syrian golden hamsters were administered radiolabeled MeHg on day 12 of gestation. The offspring was nursed by foster mothers unexposed to MeHg, while the pups from the unexposed animals were nursed by the MeHg-administered animals. Under these conditions, each pup in the litter received a dose of MeHg in utero corresponding to 0.9% of the maternal dose. The average amount of mercury found in the pups exposed via milk corresponded to 4.5% of the total body burden of the foster dam at the onset of lactation. This was about half the amount received by the pups exposed in utero. The total body burden of mercury, and the amount of mercury in the liver, brain and kidney of the pups exposed in utero began to decrease at seven days of age. The rate of decrease differed among the tissues and was lowest in the kidney. The amount of mercury in pups exposed via milk reached a peak level when the pups were 10-15 days old. The total body burden of mercury showed a slow decrease while the liver, brain and kidney levels decreased rapidly. In both groups of animals, up to 80% of the total body burden of mercury was found in the pelt. These data show that milk may be a significant exposure route for mercury and that neonatal hamsters are unable to demethylate MeHg and excrete mercury in urine and faeces.

Biological control of Botrytis cinerea growth on apples stored under modified atmospheres.

The combined effect of modified-atmosphere packaging and the application of a bacterial antagonist (Erwinia sp.) on Botrytis cinerea growth on apples (cv. 'Golden Delicious') was investigated. Inoculated apples were stored in polyethylene bags at 5 degrees C. The initial gas composition in each bag was set according to a central composite experimental design involving five levels of O2 (1 to 15%) and CO2 (0 to 15%). Control samples under ambient conditions were also included. Without the antagonist, measurements of mold colony diameter over time showed that O2 had no effect on the growth of B. cinerea, while increased CO2 levels delayed its growth by about 4 days. Application of the antagonist resulted in a significant interaction between O2 and CO2. At low O2 levels, CO2 had no effect on mold growth, but at high O2, CO2 enhanced mold growth. O2 and the antagonist worked synergistically to reduce mold growth by about 6 days at low levels of CO2. However, at high CO2 levels, O2 had no effect. The strongest antagonistic effect was observed under ambient conditions. Overall, results showed that high CO2 atmospheres can slow the growth of B. cinerea and that Erwinia sp. was an effective antagonist against B. cinerea growth on apples, particularly under ambient conditions.

The semiconductor elements arsenic and indium induce apoptosis in rat thymocytes.

Indium arsenide and gallium arsenide are important new materials in the semiconductor industry due to their superior electronic properties in comparison with the older silicon-based materials. Animal experiments have shown that exposure to these compounds induces marked alterations in gene expression and immune response. Toxicity to the immune system has frequently been related to T and B cell apoptosis. In the present study we show that the semiconductor elements indium (In) and arsenic (As) are able to induce apoptosis in rat thymocytes in vitro. The results show that exposure to InCl3 (1, 10, or 100 microM) or Na AsO2 (0.01, 0.1, or 1 microM) induced DNA laddering after 6 h of incubation without compromising cell viability. These results were corroborated by flow cytometry analysis of propidium iodide-loaded cells, showing a typical high hypodiploid DNA peak in apoptotic thymocytes. Higher doses of In (1 mM) or As (10-100 microM) induced cell death by necrosis. These data indicate that In and As can induce apoptosis and necrosis in T lymphocytes in a dose-dependent manner, which may be of relevance for their immunotoxicity.

Transplacental and lactational exposure to mercury in hamster pups after maternal administration of methyl mercury in late gestation.

Pregnant Syrian golden hamsters were given a single oral dose of 203Hg-labelled methyl mercury (MeHg), 1.6 mumol/kg body weight, on day 12 of gestation. The uptake, retention and tissue distribution of 203Hg in the dams and pups was studied by gamma-counting during the following three weeks. The average transplacental transfer of 203Hg was 1.1% of the administered dose per pup, corresponding to 11% of the administered dose to a whole litter. This was considerably more than in our previous studies when the dams were treated on gestational day 2 (1.3%) or 9 (4.6%). The amount of 203Hg transferred to each pup in utero was independent of the litter size. The average additional transfer of 203Hg to a litter via milk was 1.7% of the administered dose. In the pups, the content of 203Hg in the liver and brain decreased, while the content in the kidneys and pelt increased during the second and third week. The highest amount of 203Hg was generally found in the pelt, which indicated that unweaned hamster pups primarily excrete MeHg by binding to hair. The chemical form of mercury in the liver and kidneys of the pups was determined by ion-exchange separation of inorganic Hg and MeHg followed by gamma-counting. The amount of inorganic Hg in the liver of the pups remained constant throughout the experiment, while it increased in the kidneys after one week due to the demethylation of MeHg. The inorganic Hg in the liver of newborn pups was probably due to maternal demethylation of MeHg and transplacental transfer of inorganic Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactational exposure to methylmercury in the hamster.

Syrian Golden hamster dams were administered 203Hg-labelled methyl mercury (MeHg; 1.6 mumol/kg) 1 day after parturition and milk was collected twice during the 1st week. The excretion of 203Hg in milk and the uptake, retention and tissue distribution of 203Hg in the pups was studied using gamma counting. The fraction of inorganic Hg in milk and in the kidneys of the pups was determined following separation of inorganic Hg and MeHg by ion exchange chromatography. The concentration of 203Hg in milk on the 1st day after MeHg administration was 0.12 nmol/g. 203Hg was mainly (80-90%) excreted as MeHg during the first 6 days of lactation. The whole body and tissue concentration of 203Hg in the pups increased for 10-15 days and decreased thereafter. The content of 203Hg in the pelt and the fraction of inorganic Hg in the kidney increased throughout the study period (4 weeks). The excretion of MeHg in milk corresponded to at least 5% of the dose administered to the dam. Our study demonstrates that breast milk may be a significant source of MeHg exposure during the critical neonatal period.

Metabolism of mercury in hamster pups administered a single dose of 203Hg-labeled methyl mercury.

Golden Syrian hamster pups were administered a single subcutaneous dose of 203Hg-labeled methyl mercury (MeHg), 0.4 nmol/g body weight, seven days after birth, and were sacrificed 2, 7, 14, 21 or 28 days later. The excretion of 203Hg followed a biphasic elimination pattern with an average half-time of 8.7 days for the rapid component. The slow component had a much longer half-time and probably reflects binding of 203Hg to growing hair. The concentration of 203Hg in the liver, kidneys and brain two days after administration was 0.44, 0.38 and 0.19 nmol/g, respectively. The retention of 203Hg was higher in the kidney than in the liver and the brain. The content of inorganic 203Hg in the liver and kidneys increased the first weeks after administration, demonstrating that hamsters are able to demethylate MeHg before two weeks of age.

Demethylation and placental transfer of methyl mercury in the pregnant hamster.

The demethylation and placental transfer of methylmercury (MeHg) was studied in Syrian Golden hamsters administered a single oral dose of 203Hg-labeled MeHgCl, 1.6 mumol/kg body weight, on day 2 or 9 of gestation and sacrificed 1 day before expected parturition. In order to evaluate the role of demethylation for transplacental transport of MeHg, four hamsters were administered 203Hg-labeled HgCl2 intravenously on day 9 of gestation. The mean biological halftime of 203Hg in animals administered radiolabeled MeHg was 7.7 days and the fecal route was the main excretory pathway. The fetal content of 203Hg in hamsters administered radiolabeled MeHg on gestational day 2 or 9 corresponded to 1.3% and 4.6% of the administered dose, respectively. The distribution of 203Hg in the fetus was more even than in the dam and the concentration of 203Hg in the fetal brain, liver and kidney was similar to that of the placenta. Inorganic Hg was found in maternal liver (18% of total Hg), kidney (31%) and placenta (21%) and fetal liver (3%). The amount of inorganic 203Hg in fetal liver corresponded to about 0.015% of the dose administered to the dam as MeHg. When hamsters were administered 203HgCl2 by intravenous injection on day 9 of gestation, the concentration of 203Hg in fetal liver corresponded to 0.03% of the administered dose. The inorganic 203Hg detected in fetal liver after maternal exposure to MeHg was probably due to demethylation of MeHg in the dam and transplacental transfer of inorganic Hg.

Effect of methyl mercury exposure on the uptake of radiolabeled inorganic mercury in the brain of rabbits.

Exposure to mercuric compounds at high dose levels has previously been shown to alter the integrity and function of the blood-brain barrier in laboratory animals. In the present study, we have investigated the distribution of intravenously administered inorganic 203Hg in rabbits additionally exposed to MeHg. A single dose of 203HgCl2 was administered together with or 5 min. or 24 hr after administration of a single dose (10 or 37.5 mumol/kg b.wt.) of MeHg. In another experiment, 203HgCl2 was administered to rabbits subchronically exposed to MeHg (1 mumol/kg b.wt. daily for three weeks) 24 hr after cessation of treatment. The integrity of the blood-brain barrier was assayed by measuring the uptake of 203Hg in the brain, as the blood-brain barrier usually serves to exclude inorganic Hg from the brain. The concentration of 203Hg within the brain was similar in all MeHg-treated rabbits, corresponding to 0.02% of the administered dose, and not different from that of control animals. Under these conditions, no obvious damage to the blood-brain barrier by MeHg could be observed.

Distribution of mercury in rabbits subchronically exposed to low levels of radiolabeled methyl mercury.

The metabolism of methyl mercury (MeHg) has been studied in rabbits administered 203Hg-labeled methyl mercuric chloride, 0.125 mumol/kg body weight, twice a week for 9 weeks, by intravenous injection. Twelve weeks after cessation of treatment, about 54% of the administered dose had been excreted in faeces and 5% in urine. After one week, the highest concentration of 203Hg was found in fur (8.6 nmol Hg/g). Substantially lower concentrations were found in kidney (2.5 nmol/g), liver (0.9 nmol/g), brain (0.4 nmol/g), muscle (0.3 nmol/g) and blood (0.1 nmol/g). The rate of elimination of 203Hg from brain, muscle and blood was faster (t1/2 about 12 days) than that from kidney and liver (t1/2 about 28 days). The relative amount of inorganic Hg in kidney and liver increased with time after cessation of treatment. The highest fractions were 85 and 70%, respectively. In brain, no significant demethylation of MeHg could be detected.

Effects of glutathione transferase activity on benzo[a]pyrene 7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay.

This study deals with the role of glutathione transferase (GST)-mediated conjugation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) in two mammalian cell lines, human mammary carcinoma cells (MCF-7) and rat hepatoma cells (H4IIE), in relation to their capacity to metabolize (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)-BP-7,8-diol] to products that induce mutations in co-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized (-)-BP-7,8-diol to BPDE, but mutations in co-cultivated V79 cells were only detected with MCF-7 cells. However, depletion of glutathione (GSH) in H4IIE cells increased the mutagenicity of (-)-BP-7,8-diol to a similar level to that found with MCF-7 cells. Measurements of GST activity using GSH and post-microsomal supernatants from H4IIE, V79 and MCF-7 cells indicated a substantial difference in conjugation capacity. Although preparations from all three cell-lines showed GST activity with 1-chloro-2,4-dinitrobenzene as the substrate, GST activity towards BPDE could only be detected in supernatants from H4IIE cells. This is consistent with the presence of GST 7-7 an isoenzyme highly efficient in catalysing BPDE-GSH conjugation. The difference in GSH-conjugation activity towards BPDE was confirmed using intact H4IIE and MCF-7 cells in culture. These results indicate that GSH-conjugation plays a pivotal role in mutagenesis induced by polycyclic aromatic hydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugation capacity may be regarded as one important factor in defining a target cell population with an increased risk for tumour initiation following exposure to PAH.

Metabolism of methylmercury in rabbits and hamsters.

The metabolism of 203Hg-labeled methylmercury chloride (MeHg) has been studied in rabbits and hamsters. Rabbits were administered 1.6 mumols MeHgCl/kg bw intravenously, and hamsters 40 mumols/kg bw orally. Urine and feces were collected daily and groups of four animals killed after 1 h, 1 d, or 7 d. The concentration of 203Hg in blood, liver, kidney, spleen, lung, heart, and brain was determined by gamma counting. In both animal species, the clearance of 203Hg in the brain was slower than in other tissues. In the rabbits the brain 203Hg concentration increased during the whole experimental period. Rabbits excreted 203Hg primarily in feces (about 20% of the dose within 1 wk), and much less in urine (less than 2%). In contrast, hamsters very efficiently excreted 203Hg in urine (50% in 1 wk). The fecal excretion was similar to that of the rabbits. Separation of inorganic Hg and MeHg in urine from hamsters by ion exchange chromatography showed that about 90% of the urinary 203Hg was excreted as MeHg. These findings show that rabbits and hamsters are interesting experimental animal systems for studying the metabolism of MeHg.

Induction of rat liver glutathione transferase isoenzyme 7-7 by lead nitrate.

The glutathione transferase (GST) activity of rat liver cytosolic preparations with ethacrynic acid (EA) and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene (BPDE) as substrates, increased by 125 and 350%, respectively, in animals that had been treated with a single intravenous dose of Pb(NO3)2 (100 mumol/kg body wt) 48 h prior to sacrifice, whereas activity with 1-chloro-2,4-dinitrobenzene (CDNB) increased only about 60%. No induction of these activities was observed in cytosolic preparations from regenerating rat liver, whereas cytosols prepared from hepatocyte nodules showed increased activity with all three substrates (EA: 400%; BPDE: 790%; CDNB: 205%). These results suggest that Pb(NO3)2 is an inducer of GST 7-7, an isoenzyme that has been associated with hepatocarcinogenesis. Elucidation of the mechanism of GST 7-7 induction by lead may contribute to our understanding of the process of chemical carcinogenesis.

The influence of cytochrome P-450 induction on the disposition of carcinogenic benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide in isolated cells.

The disposition of the carcinogenic (+)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] has been studied in isolated hepatocytes obtained from 3-methylcholanthrene-pretreated rats. In these cells different routes are acting in concert and contribute to diol-epoxide elimination. Conjugation of (+)-anti-BPDE with glutathione (GSH) and cytochrome P-450c-mediated metabolism of the diol-epoxide to 1- and 3-hydroxy-anti-BPDE (triol-epoxides) appears to be equally important. The reactive triol-epoxides undergo a number of secondary reactions, including covalent binding to cellular constituents, e.g. protein and GSH, and hydrolysis to pentahydroxyderivatives. The effective intracellular lifetime of (+)-anti-BPDE is approximately 1 min and comparable to that previously observed in hepatocytes obtained from uninduced animals.

Glutathione transferase catalyzed conjugation of benzo[a]pyrene 7,8-diol 9,10-epoxide with glutathione in human skin.

Glutathione transferase (GST) activity towards racemic as well as the resolved enantiomers of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (anti-BPDE) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in post-microsomal supernatants (PMS) obtained from eight human skin samples. All preparations showed significant activity towards anti-BPDE and an almost exclusive preference for the more tumourigenic (+)-enantiomer. The specific activity towards (+)-anti-BPDE varied about five-fold between different PMS (range 147-781 pmol/min per mg protein) whereas the variation in specific activities towards CDNB was about two-fold (range 30-71 nmol/min per mg protein). The activities obtained with PMS at saturating concentrations of racemic anti-BPDE were about half of the activity towards the (+)-enantiomer indicating that (-)-anti-BPDE competitively inhibits conjugation of the (+)-form. No correlation was evident between the activities towards (+)-anti-BPDE and CDNB implying that different classes of GST isoenzymes participated in the two different reactions. Immunoblot analysis revealed the presence of Class Alpha and Pi isoenzymes whereas Class Mu isoenzymes seemed to be absent in the human skin samples analyzed. Quantitatively, the Class Pi isoenzyme(s) predominated in all skin samples and the amount of enzyme was about 1-3 micrograms GST Pi/mg PMS protein. The almost exclusive conjugation of (+)-anti-BPDE by PMS and previous results with GST Pi enzymes from human placenta suggested that this type of enzymes catalysed the conjugation reaction. The five-fold variation in specific activity towards (+)-anti-BPDE observed among the different PMS may be explained by individual differences in GST Pi content or by the presence of endogenous modifiers of GST activity towards the diol-epoxide.