Growth inhibitory and biocidal activity of some isothiazolone biocides

C ollier , P.J., R amsey , A.J., A ustin , P. & G ilbert , P. 1990. Growth inhibitory and biocidal activity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 569–577.
Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizo-saccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1–0.5 μg/ml for CMIT, 15–20 μg/ml for BIT and 40–250 μg/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this com-pound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Chemical reactivity of some isothiazolone biocides

C ollier , P.J., R amsey , A., W aigh , R.D., D ouglas , K.T., A ustin , P. & G ilbert , P. 1990. Chemical reactivity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 578–584.
Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7–10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus]

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.     

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

Naegleria fowleri and N. lovaniensis: differences in sensitivity to trimethoprim and other antifolates

The growth of Naegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 micrograms/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 micrograms/ml. N. lovaniensis propagation in the same medium was inhibited with 10 micrograms/ml of trimethoprim, 50 micrograms/ml methotrexate and 100 micrograms/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 micrograms/ml. The inhibitory effect of trimethoprim on N. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killed Enterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tetrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity in N. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate of N. fowleri amoebae did not influence the trimethoprim inhibition of N. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation for N. fowleri antifolate resistance.     

Mast cell stabilizing and lipoxygenase inhibitory activity of Cedrus deodara (Roxb.) Loud. wood oil

Volatile oil of C. deodara, administered orally at the doses of 50, 100 and 200 mg/kg body weight, significantly inhibited the pedal edema induced by compound 48/80 in rats. The oil significantly inhibited compound 48/80 induced degranulation of isolated rat peritoneal mast cells at concentrations ranging from 25-200 micrograms/ml. C. deodara wood oil also significantly inhibited the enzyme lipoxygenase at a concentration of 200 micrograms/ml. Thus, the anti-inflammatory activity of C. deodara wood oil could be attributed to its mast cell stabilizing activity and the inhibition of leukotriene synthesis.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

A potent allelopathic substance in cucumber plants and allelopathy of cucumber

A potent growth inhibitory substance was isolated from an aqueous methanol extract of cucumber (Cucumis sativus L. cv. Phung Tuong) plants and determined as (2S)-2,3-dihydro-2??-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-(1,2,3-trihydroxypropyl)benzofuran-3??-methanol (sisymbrifolin) by spectral data. Sisymbrifolin inhibited the growth of cress (Lepidium sativum) and Echinochloa crus-galli seedlings at concentrations greater than 3???M. Concentration of sisymbrifolin in the cucumber plants was the greatest among four growth inhibitory substances, (S)-2-benzoyloxy-3-phenyl-1-propanol, 9-hydroxy-4,7-megastigmadien-9-one, (6S,7E,9S)-6,9,10-trihydroxy-4,7-megastigmadien-3-one, and sisymbrifolin found in the cucumber, whereas growth inhibitory activity of 9-hydroxy-4,7-megastigmadien-9-one against cress and E. crus-galli was the greatest. Total activities of these substances (concentration of the substance/concentration required 50?% growth inhibition) were 14.4, 13.2, 8.5 and 10.7 for (S)-2-benzoyloxy-3-phenyl-1-propanol, 9-hydroxy-4,7-megastigmadien-9-one, (6S,7E,9S)-6,9,10-trihydroxy-4,7-megastigmadien-3-one and sisymbrifolin, respectively. These total activities were about 100-fold greater than those of phenolic acids, which are often mentioned as putative allelochemicals of plants. Thus, these substances may play important roles in the allelopathy of cucumber plants through the growth inhibition of neighboring plant species.     

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 micrograms/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < or = 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 micrograms/ml) and B. subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively. Both compounds presented MIC of 3.12 micrograms/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 micrograms/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.     

Effect of unsaturated fatty acids in growth inhibition of some penicillin-resistant and sensitive bacteria

The growth of two penicillin-resistant Gram-positive bacteria, Bacillus licheniformis (749/C, penicillin G-resistant) and Staphylococcus aureus (metR 18, methicillin-resistant) and one Gram-negative strain, Escherichia coli (cloxacillin-resistant) as well as that of their wild counterparts was inhibited by the long-chain unsaturated fatty acids, linoleic, linolenic and arachidonic acid. The minimum inhibitory concentrations (MIC) of all the fatty acids were found to be 4-6 micrograms/ml for Staph. aureus (metR 18 & wild), 8-30 micrograms/ml for B. licheniformis (749/C & wild) and 70-90 micrograms/ml for E. coli (cloxacillin-resistant & wild). The inhibitory activity increased as the number of double bonds in the fatty acids increased. In most instances the concentrations of fatty acids required to inhibit the growth of the penicillin-resistant strains were lower than that required for their sensitive counterparts. This inhibition of growth in the presence of fatty acids may be due to an increase in permeability of the membrane as evidenced by the measurement of the leakage of 260 nm absorbing material and fluidity.     

Chalcones and flavonoids as anti-tuberculosis agents

A series of flavonoids, chalcones and chalcone-like compounds were evaluated for inhibitory activity against Mycobacterium tuberculosis H37Rv. Among them, eight compounds exhibited >90% inhibition on the growth of the bacteria at a concentration of 12.5 microg/mL. Chalcones 1-(2-hydroxyphenyl)-3-(3-chlorophenyl)-2-propen-1-one (22) and 1-(2-hydroxyphenyl)-3-(3-iodophenyl)-2-propen-1-one (37) demonstrated 90 and 92% inhibition, respectively. Chalcone-like compounds (heterocyclic ring-substituted 2-propen-1-one) 1-(4-fluorophenyl)-3-(pyridin-3-yl)-2-propen-1-one (48), 1-(3-hydroxyphenyl)-3-(phenanthren-9-yl)-2-propen-1-one (49), 1-(pyridin-3-yl)-3-(phenanthen-9-yl)-2-propen-1-one (50) and 1-(furan-2-yl)-3-phenyl-2-propen-1-one (51) exhibited 98, 97, 96 and 96% inhibition, respectively. The actual minimum inhibitory concentrations (MIC), defined as the lowest concentration inhibiting 99% of the inoculum, for 22, 37, 48, 49, 50 and 51 were 20.3, 31.5, 48.3, >35.7, 6.8 and 19.2, respectively. A hydrophobic substituent on one aromatic ring, and a hydrogen-bonding group on the other aromatic ring resulted in increased anti-TB activity of the chalcones and chalcone-like compounds. Flavones and flavanones are more geometrically constrained than the corresponding chalcone analogues. The decreased activity of the flavones with respect to the chalcones may be due to the confinement of the terminal aromatic rings to the same plane.     

Antimicrobial activity of 2-aminophenoxazin-3-one under anaerobic conditions.

Potential toxicity of 2-aminophenoxazin-3-one to 20 bacterial species and two fungi and its inactivation under anaerobic conditions were investigated. Minimum inhibitory concentration for cellulolytic bacteria was in the range of 50-100 micrograms.mL-1, but at 100 micrograms.mL-1 of 2-aminophenoxazin-3-one, there was no effect on the growth of any of the noncellulolytic bacteria. Four noncellulolytic bacterial strains showed no inhibition of growth, even at 200 micrograms.mL-1 of this compound. Under anaerobic conditions and in the presence of cysteine, the long wavelength absorption band of this compound slowly shifted from about 434 to 320 nm, and its inhibitory effect on RNA synthesis was relieved after one-half of a generation time in cultures of cellulolytic ruminal bacteria. A similar shift of absorption band was observed in rumen fluid filtered through a 0.22-microns Millipore filter. It was concluded that protonation of 2-aminophenoxazin-3-one under anaerobic conditions present in the rumen would considerably reduce its potential toxicity to cellulolytic bacteria.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Lysosomal enzyme release from human monocytes by particulate activators is mediated by beta-glucan inhibitable receptors

Human peripheral blood monocytes ingest particulate activators and generate leukotrienes via a trypsin-sensitive, beta-glucan-inhibitable receptor. The incubation of monolayers of monocytes with from 4 X 10(5) to 2 X 10(8) zymosan or glucan particles resulted in a dose-dependent release of up to 9% +/- 1.9 and 17.8% +/- 5.3 (mean +/- SD, n = 3) of the lysosomal enzyme, N-acetylglucosaminidase, into the culture medium. Lysosomal enzyme release occurred throughout the 2-hr period studied, with the greatest rate of N-acetyl-glucosaminidase release occurring during the first hour; the presence of 5 micrograms/ml of cytochalasin B accelerated this process when zymosan was the agonist. The preincubation of monocytes with from 0.5 to 500 micrograms/ml of soluble yeast beta-glucan inhibited N-acetylglucosaminidase release by 4 X 10(7) zymosan and glucan particles in a dose-dependent manner, with 50% inhibition occurring with 50 micrograms/ml of soluble yeast beta-glucan (mean +/- SD, n = 3). Preincubation with as much as 5 mg/ml of yeast mannan had no inhibitory effect on N-acetylglucosaminidase release. The pretreatment for 30 min of monolayers of monocytes with 50 micrograms/ml of affinity-purified trypsin, which selectively inactivates the monocyte-phagocytic response to particulate activators, also fully inhibited lysosomal enzyme release induced by zymosan and glucan particles. The inhibitory effects of a soluble ligand, yeast beta-glucan, and of trypsin pretreatment on lysosomal enzyme release correspond to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles and thus indicates ligand specificity for the beta-glucan receptor in the release of stored intracellular mediators.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Estradiol-17 beta inhibition of lutropin and dibutyryl cyclic AMP induced steroidogenesis in intact bovine luteal cells. Absence of effect on induced protein phosphorylations

Estradiol-17 beta is known to inhibit in a dose dependent manner the lutropin-induced stimulation of progesterone synthesis in luteal cells without affecting the intracellular cyclic AMP increase produced by the hormone. The hypothesis that this inhibitory action could involve an inhibition of the cyclic AMP dependent phosphorylation of cytosolic proteins was investigated by using incubations of selected small bovine luteal cells. Doses of 10 and 100 micrograms/ml of estradiol-17 beta inhibited respectively 60 and 90% of the progesterone synthesis induced by lutropin as well as by dibutyryl cyclic AMP in small bovine luteal cells. At the concentrations of 10 and 100 micrograms/ml, estradiol-17 beta was unable to affect the cyclic AMP dependent protein kinase activation induced by lutropin. At the concentration of 10 micrograms/ml the steroid was without effect on the lutropin or dibutyryl cyclic AMP induced protein phosphorylations. However 100 micrograms/ml of estradiol-17 beta seemed to produce a slight inhibition of the induced protein phosphorylations.