Sensitivity of salmonella YG5161 for detecting PAH‐associated mutagenicity in air particulate matter

The Salmonella/microsome assay is the most used assay for the evaluation of air particulate matter (PM) mutagenicity and a positive correlation between strain TA98 responses and benzo[a]pyrene (B[a]P) levels in PM has been found. However, it seems that the major causes of PM mutagenicity in this assay are the nitro and oxy‐PAHs. Salmonella YG5161, a 30‐times more responsive strain to B[a]P has been developed. To verify if YG5161 strain was sufficiently sensitive to detect mutagenicity associated with B[a]P mutagenicity, PM samples were collected in Brazil and Sweden, extracted with toluene and tested in the Salmonella/microsome microsuspension assay. PAHs and B[a]P were determined and the extracts were tested with YG5161 and its parental strain TA1538. The extracts were also tested with YG1041 and its parental strain TA98. For sensitivity comparisons, we tested B[a]P and 1‐nitropyrene (1‐NP) using the same conditions. The minimal effective dose of B[a]P was 155 ng/plate for TA1538 and 7 ng/plate for YG5161. Although the maximum tested dose, 10 m³/plate containing 9 ng of B[a]P in the case of Brazilian sample, was sufficient to elicit a response in YG5161, mutagenicity was detected at a dose as low as 1 m³/plate (0.9 ng). This is probably caused by nitro‐compounds that have been shown to be even more potent than B[a]P for YG5161. It seems that the mutagenicity of B[a]P present in PM is not detectable even with the use of YG5161 unless more efficient separation to remove the nitro‐compounds from the PAH extract is performed. Environ. Mol. Mutagen. 55:510–517, 2014. © 2014 Wiley Periodicals, Inc.     

Mutagenic activity of carbon black dyes used in the leather industry

Seven carbon black pastes used as commercial leather dyes weretested for their mutagenicity in the Salmonella/microsome test(TA98 and TA100 strains). All the samples assayed either directlyor after extraction with a 30-min sonication in benzene weredevoid of mutagenicity both in the presence and absence of ametabolic activation preparation. After a 48-h extraction withboiling toluene in a Soxhlet apparatus, four samples were mutagenicin TA98 strain in the presence of S9 mix. The activity rangedfrom 1.3 to 9.6 induced revertants/mg equivalent of extract.A weak direct mutagenic activity in strain TA98 was shown byone extract. Polycyclic aromatic hydrocarbons (PAH) were determinedin the toluene extracts by high resolution gas chromatography/massspectrometry. The presence of PAH could explain the mutagenicityof only one sample (8.79 µg of total PAH/100 mg equivalentsof extract), while low or undetectable levels of PAH were foundin the other mutagenic extracts. The mutagenic activity wasevident only after a vigorous extraction process, thus a lowbioavailability of the mutagens present in these compounds issuggested. ²To whom correspondence should be addressed     

Alteration of glucose-6-phosphatase activity and regenerative, and total DNA concentrations in regenerating and normal rat liver of aqueous extracts of two Nigerian plants

The effect(s) of an intraperitoneal (i.p.) administration of aqueous extracts of two Nigerian medicinal plants, Garcina cola and Vermonia amygdalina on the level of glucose-6-phosphatase activity, total protein content and regenerative and total DNA concentrations, was determined in liver homogenates of partially-hepatectomized and normal albino rat liver. The established control curves for the DNA and microsome syntheses rates after partial hepatectomy showed two waves of synthetic activity; the first occurring at 24 hrs., and the second at 32 hrs as judged by total regenerative DNA concentration and glucose-6-phosphatase activity. Garcina cola significantly inhibited the first wave of microsome and DNA syntheses by 31% and 24% respectively in a dose-related manner. However, the degree of inhibition of glucose-6-phosphatase activity by Garcina cola was substantially reduced in normal rat liver when administered simultaneously with carbon tetrachloride. Dilute and concentrated extracts of Vermonia amygdalina, on the other hand, elevated the levels of glucose-6-phosphatase activity in normal rat liver by 44% and 54%, respectively over the controls. Total DNA concentration was similarly increased, though to a lesser degree. The implication of these results are discussed in relation to the structural similarity of the furocoumarins contained in these plants to AFB1, and their likely modes of action.     

Lack of genotoxicity of silver iodide in the SCE assay in vitro, in vivo, and in the Ames/microsome test

Silver iodide was evaluated for mutagenicity in the Ames/microsome test (strains TA 1535, TA 102, TA 97, and TA 98) and for the ability to induce Sister Chromatid Exchanges (SCE) in human cultured lymphocytes and in P388 lymphocytic leukemia cells cultured in the mouse peritoneal cavity. From the cytogenetic in vitro studies, it was observed that silver iodide, either in acetone solutions or as a suspension with polyacrilamide, scarcely causes a doubling effect on SCEs at nearly toxic concentrations (1 microg/ml). Such a doubling effect by silver iodide on SCEs in P388 leukemia cells in vivo was not achieved even after using 100 microg/g mouse body weight. In the Ames/microsome test actually a doubling effect on revertants was only isolately achieved with 30 microg/ml in TA 102 (S9-) and at 150 microg/ml in TA 97 (S9+) doses, which appear to be nearly toxic for bacteria.     

Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.     

Use of indomethacin to demonstrate enterotoxic activity in extracts of Entamoeba histolytica trophozoites.

The present study was designed to develop and characterize animal models for the assay of enterotoxic activity in extracts of Entamoeba histolytica trophozoites. Marked water and electrolyte secretion occurred in both in vivo rabbit ileal loops and rat colon loops exposed to clarified sonic fluids of E. histolytica strain HM-1 trophozoites (10(6)/ml) when the animals were first administered indomethacin (0.1 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg) dose of indomethacin. Higher doses (greater than or equal to 1 mg/kg) of indomethacin inhibited extract-induced secretion. No enterotoxic activity was detected with or without indomethacin, using extracts from the nonpathogenic E. histolytica-like Laredo strain, even at 10-fold-higher cell concentrations. The HM-1 enterotoxic activity was heat labile. Prior exposure of the loop lumen to fetuin (100 micrograms/ml) blocked the secretory response to subsequent HM-1 extract exposure, but postexposure of the loop to fetuin did not block secretion that had already been established by the amoeba extract. No histological changes were seen associated with the amoeba extract-induced secretion. The data suggest that E. histolytica HM-1 strain elaborates an enterotoxic activity capable of causing consistent secretion in the mammalian intestine that has had its mucosal cytoprotection impaired by indomethacin.     

Isolation and immunochemical properties of burn toxin.

Human skin and the skin of some laboratory animals was minced, scalded and homogenized. The preparation from scalded human skin (the "crude burn toxin") revealed to be highly toxic to mice after i.v. administration. It was found by immunodiffusion and immunoelectrophoresis, that the urea and buthanol extracts of the crude "toxin" contain tissue antigens which were absent in a control preparation from a native (non scalded) skin.     

Oral toxicities of Clostridium botulinum type A and B toxins from different strains

The production and the oral toxicity for mice of Clostridium botulinum type A and B toxins of different strains were studied. All five type B strains produced both 16S (large or L) and 12S (medium or M) toxins, although the relative amounts varied with the strains. The culture supernatant of type B Okra strain was the most potent in oral toxicity. The L toxin of this culture was about 700 times more toxic in feeding tests with mice than the L toxin from type B strain NH-2, whereas the M toxins of the two strains had the same oral toxicity. These results indicate that the oral toxicity of type B toxin varies with the culture strain. Oral toxicities of L toxin produced by type A strains 62A and 97 were comparable but were 10 times higher than those of their M toxins. Hybrids of toxic and nontoxic components separated from L toxins of type B strains Okra and NH-2 revealed that the high oral toxicity of the B-L toxin of strain Okra is attributable not to the toxic but to the nontoxic component of the toxin. The present study suggests that the 16S molecular-sized toxin elaborated by a certain strain of C. botulinum type B is implicated in the high fatality rate in type B human botulism.     

Toxicity of lipopolysaccharide and of soluble extracts of Salmonella typhimurium in mice immunized with a live attenuated aroA salmonella vaccine.

Mice immunized intravenously 10 days earlier (but not those immunized 2 months earlier) with an attenuated Salmonella typhimurium SL3261 aroA live vaccine and tested for delayed-type hypersensitivity by injection of crude Salmonella extracts in the footpad can die within 24 to 48 h of an unexplained allergic reaction. The lethal reaction could be prevented by prior administration of anti-tumor necrosis factor alpha serum. Injection of lipopolysaccharide (LPS) (either purified phenol-water-extracted [Westphal] LPS or protein-rich trichloracetic acid-extracted [Boivin] LPS) was also lethal for mice immunized 10 days before. An LPS-rich crude Salmonella extract was more toxic than one which contained less LPS, suggesting that LPS may have been involved in the lethal reactions to crude antigens. Mild alkaline hydrolysis removes O-linked acyl groups from lipid A and eliminates many toxic effects of LPS; however, both Boivin LPS and Westphal LPS remained toxic for immunized mice after alkaline hydrolysis. In contrast, alkaline hydrolysis of crude whole Salmonella extracts (which caused marked protein degradation) reduced the lethal toxicity of the extracts, especially for an LPS-rich preparation. Mice immunized orally with the live vaccine did not show hypersensitivity to either LPS or crude extracts. The results suggest that the lethal reaction to crude Salmonella antigens in mice immunized 10 days earlier is complex, that tumor necrosis factor alpha is involved, and that allergic reactions to crude antigens (but not to LPS alone) can be reduced by mild alkaline hydrolysis.     

Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus.

The microbicidal activities of freeze-thaw and high-salt extracts of human and bovine polymorphonuclear leukocyte (PMN) granules were tested against a smooth intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus which differ in virulence and survival within PMNs. Freeze-thaw extracts of human PMN granules were more brucellacidal than high-salt extracts when supplemented with hydrogen peroxide (H2O2) and potassium iodide (KI), whereas the opposite was found with freeze-thaw and high-salt extracts of bovine PMN granules. There was no oxygen-independent killing of either the smooth or rough strain of B. abortus by amounts of granule extracts which caused 100% killing of a deep rough mutant (Re) of Salmonella typhimurium. The oxygen-dependent brucellacidal activity of granule extracts was dependent on concentrations of myeloperoxidase (MPO) units, H2O2, and KI. Maximal brucellacidal activity was observed at pH 5.5 to 6.0. The smooth strain, 45/0, was more resistant to oxygen-dependent killing by granule extracts than was the rough strain, 45/20. Granule extracts were more brucellacidal than purified MPO at equivalent levels of MPO enzyme units, suggesting that at least one other reaction enhances killing by the MPO-H2O2-I- system.     

Immunochemical detection of aflatoxigenic potential of Aspergillus species with antisera prepared against enzymes specific to aflatoxin biosynthesis

Antisera against a methyltransferase specific to the late stages of the aflatoxin biosyn‐thetic pathway were produced in rabbits and were characterized by double diffusion, immunoelectrophoretic analysis (IEA), and crossed‐IEA. Analysis of a homogeneous preparation of the enzyme (purity>95%) with the antisera revealed two antigenic components. The presence of specific methyltransferase in the extracts of several Aspergillus spp. was ascertained by measuring the continuity of identical precipitin lines between adjoining patterns in the line‐IEA. The precipitin lines, typical for the purified enzyme, were also observed in the extracts of several Aspergillus flavus and A. parasiticus strains that had methyltransferase activity; but no reactivity was detected in extracts of other Aspergillus, Fusarium and Penicillium spp. that did not exhibit the enzyme activity. The present study indicates that an immunochemical profile for the methyltransferase could be used as a tool in the identification of the aflatoxigenic potential of various Aspergillus spp.     

Production of an extracellular toxin by the oral pathogen Campylobacter rectus.

The ATCC type strain and six clinical isolates of Campylobacter rectus were tested for toxicity against HL-60 cells and human polymorphonuclear neutrophils (PMNs). After challenge with bacterial cell suspensions and media supernatants for up to 4 h, eukaryotic cell viability was assayed by trypan blue dye exclusion and lactate dehydrogenase release. Cells of the C. rectus type strain were not toxic. However, ethanol and (NH4)2SO4 extracts of culture media supernatants killed HL-60 cells in a time and dose dependent manner with 700 micrograms of supernatant protein killing 100% of HL-60 cells in 4 h. Concentrated media supernatants from clinical isolates also killed 100% of HL-60 cells in 30 to 60 min. The bacterial culture supernatants were toxic to PMNs with clinical isolates killing 70 to 90% of PMNs in 2 to 4 h. SDS-PAGE and immunoblot analysis of the toxic media supernatants revealed C. rectus specific proteins and lipopolysaccharide (LPS). The toxic activity was inhibited by protease, indicating that the toxin was protein. Non-toxic and toxic media supernatants were obtained by altering hemin and fumarate in the growth media. SDS-PAGE analysis of these revealed that all toxic supernatants contained a 104 kDa protein.     

Degradation of phenolic compounds by the yeast Candida tropicalis HP 15. II. Some properties of the first two enzymes of the degradation pathway

The first two enzymes of the phenol degradation pathway were determined and characterized in crude extracts from Candida tropicalis HP 15. The phenol hydroxylase (EC 1.14.13.7) was a stable NADPH2- and FAD-dependent enzyme with a pH-optimum of 7.6 to 8.0 and a broad substrate specificity. Influence of ultrasound rapidly reduced the enzyme activity. The catechol 1,2-oxygenase (EC 1.13.1.1) had a broad pH-optimum between 7.5 and 9.6 and a limited substrate specificity. Two active protein bands indicating the presence of two isofunctional enzymes were detectable after electrophoretic separation of crude and partially purified extracts on polyacrylamide gels and specific staining for catechol 1,2-oxygenase activity.     

Intracellular bacteria: the origin of dinoflagellate toxicity.

Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.     

Acute Toxicity Study and Antipyretic Effect of the Brown Alga Turbinaria Conoides (J. Agardh) Kuetz

The active principles of brown alga, Turbinaria conoides (J.Agardh) Kuetz. (Sargassaceae) was extracted with n-hexane, cyclohexane, methanol and ethanol-water (1:1) and investigated for acute toxicity and antipyretic activity. Phytochemical analysis of the extracts revealed the presence of steroids, flavonoids and reducing sugars. Acute toxicity study was performed in Wistar rats after administration of extracts orally. No mortality was observed up to the dose of 5g/kg for methanol and ethanol-water (1:1) extracts whereas n-hexane and cyclohexane extracts were found to be toxic at the dose levels of 1g/kg and 2 g/kg respectively. In biochemical analysis, n-hexane, cyclohexane and ethanol-water (1:1) extracts caused a significant (P<0.01) increase in serum cholesterol, protein and alkaline phosphatase levels. In haematological studies, a significant difference was observed for cyclohexane and ethanol-water (1:1) extracts in polymorphs, lymphocytes and eosinophils when compared to the control. Antipyretic activity of extracts (100–400 mg/kg doses) was carried out on yeast-induced pyrexia in rats. Cyclohexane extract exhibited more significant antipyretic activity (P<0.01) than the other extracts at a dose of 200mg/kg (54.43%), which was comparable to that of paracetamol at a dose of 33 mg/kg. The findings validated the use of this brown alga in traditional cure of children''s fever.     

Lymphocyte stimulation by murine derived renal antigens: Lymphocyte response to renal antigens

Peripheral blood lymphocytes from C57B1/6 and A/J mice were reacted in vitro with protein extracts prepared from kidneys and spleens of each strain. The magnitude of the responses, as determined by the incorporation of tritiated thymidine into DNA, was highest and most significant (P < 0.005) for allogeneic kidney extracts. When cultured with syngeneic kidney extracts, there was little or no stimulation. The response to allogeneic spleen extracts was considerably lower, and in syngeneic spleen extracts, sometimes inhibitory. These proliferation responses could be detected after 48 h of the culture period. Murine-derived antigens may prove useful as initiators of the blastogenic response and may be a valuable tool in appraisal of immunocompetence, since results can be obtained in less than 3 days.     

Amphotericin B resistance of Aspergillus terreus in a murine model of disseminated aspergillosis

The in-vivo activity of amphotericin B and itraconazole against a clinical isolate of Aspergillus terreus was determined in a murine model of disseminated aspergillosis. MICs of amphotericin B and itraconazole for the strain, determined by an NCCLS-based technique, were 2 microg/ml and 1 microg/ml, respectively. Mice infected intravenously were treated with either itraconazole (50 or 100 mg/kg/day) or amphotericin B 4.5 mg/kg/day for 10 days. Treatment with both doses of itraconazole significantly prolonged the survival rates compared with those for untreated mice. In comparison, mortality rate and median survival time were identical for mice treated with amphotericin B and for mice given no therapy, indicating that the strain was highly resistant to amphotericin B in this model. Analysis of sterol composition showed that the major sterol was ergosterol. This suggests that amphotericin B resistance was not related to a modified sterol profile.     

Modulatory Effect of the Pesticide Captan on the Immune Response in Rats and Mice

The effect of short-term oral administration of captan, [N-(trirnethylthio)-4-cyclohexene-1,2-carboximidel on the immune response was studied in rats and mice. Animals were fed a diet with or without 0.3 % (w/w) of captan for 7, 14, 21 and 42 days. The SRBC-antibody formation was depressed by about 70 % in both species after 14 days of treatment. A release of inhibition occurred in mice at day 42. In a parallel manner, the lymphoblastic stimulation of splenic cells by PHA and by LPS was studied in captan-treated mice and their controls. The stimulation by PHA of splenic cells that were mainly T cells was clearly inhibited (45 %) by day 14 of captan ingestion. Thereafter, the inhibition was only partially released until day 42. B cells, stimulated by LPS, presented a decrease in stimulation in captan-treated mice, during the first week of diet (20 %). Then an important increase in the stimulation of these cells occurred at day 21 (85 %) followed by a return to the normal value at day 42. These results pointed out a clear depressive effect of captan-diet on the immune response of the animals. The inhibition of SRBC-antibody formation during the first stage of the treatment may be correlated to the inhibitory effect of captan on T cells, which were cooperative with B cells for the expression of SRBC-antibody synthesis. These effects were obtained at a level of captan which was considerably lower than the toxic dose.     

A new method of allergen standardization. Passive cutaneous anaphylaxis inhibition in a mouse-to-rat system in comparison with other methods.

Various methods have been employed for standardization of potency of allergen extracts. We used passive cutaneous anaphylaxis (PCA) inhibition in a mouse-to-rat system as a new means of standardization. Several allergen extracts, including mite, short ragweed, house dust, Aspergillus, Candida, and Japanese cedar, were examined. Mouse IgE antibodies were produced after two or three injections of antigen and alum. The potency measured by PCA inhibition in the mouse-to-rat system was compared with that of protein nitrogen (PN) content, end point of prick test in humans, and 50% radioallergosorbent test (RAST) inhibition. Our studies suggest that the mouse-to-rat system could be used to determine allergenic potency and to standardize allergen extracts.     

Key enzymes for the degradation of benzoate,m- and p-hydroxybenzoate by some members of the order Actinomycetales

A preliminary screening of numerous species of the order Actinomycetales, especially of the genera Mycobacterium, Nocardia, Rhodococcus, Pseudonocardia, and Streptomyces, showed that many of them are able to metabolize benzoate (B) and p-hydroxybenzoate (pHB) as indicated by growth and change of color of the pH-indicator of an agar medium. Subsequent experiments with liquid cultures which allowed the analysis of substrate utilization by thin layer chromatography confirmed these results. The study of the degradative pathway proved that B was metabolized via catechol (C), pHB via protocatechuate (P) and m-hydroxybenzoate (mHB) via gentisate (G). The aromatic ring of C and P was subjected to an ortho-cleavage; only one strain of Noc. asteroides degraded C via a meta-cleavage, but P via an ortho-cleavage. Cell free extracts of four selected organisms exhibited activity of C-1,2-dioxygenase (C-1,2-O) and/or P-3,4-dioxygenase (P-3,4-O), depending on the growth substrate used for precultivation. In Streptomyces C-1,2-O was only found in cells grown on B, and P-3,4-O only in cells grown on pHB. On the contrary, in Rhodococcus rhodochrous B-cells oxidized C as well as P, while P-cells possessed only P-3,4-O-activity.