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Placental and fetal tissues obtained from humans, monkeys, rats and rabbits contained monooxygenases capable of catalyzing the formation of catechol estrogens. Treatments of pregnant rats with phenobarbital, Aroclor 1254, or 3-methylcholanthrene each increased measured rates of catechol estrogen formation in placentas, fetal brains and fetal livers, while other rat tissues exhibited either increased or decreased activity. Treatment of pregnant rabbits with Arocolor 1254 produced an increase in catechol estrogen formation in all fetal tissues studied and in the maternal liver and kidney. Catechol estrogen formation in placental microsomes of macaque monkeys (Macaca arctoides) was generally less than that observed in human placental microsomes. Human placentas obtained from smokers exhibited enhanced catechol estrogen formation and this activity appeared to be highly correlated with placental aryl hydrocarbon hydroxylase activity. The data suggested [a]pyrene in a similar fashion in placental tissues and that the enzyme systems involved may be under similar regulatory control.

Hydroxylation of oestrogens in the 2 or 4 positions leads to the formation of catechol oestrogens. These compounds are physiologically active in animals, especially in the control of gonadotropin secretion. Physiological activity can be ascribed to either an oestrogenic action or interaction with the catecholaminergic systems. These compounds are also formed in human subjects. Surprisingly high levels are found in the urine. The peri-ovulatory peak in urinary catechol oestrogen excretion is compatible with a role for these compounds in the control of gonadotropin secretion.

The correlation between the colicine resistance of the reference and isolated strains of enterobacteria and their capacity for the biosynthesis of enterocheline, as well as the influence of exogenous enterocheline on the colicine sensitivity of enterobacteria were studied. In the wild strains of enterobacteria sensitivity to colicines was shown to have no correlation with capacity for the accumulation of catechol-type sideriphores. In some cases exogenous enterocheline prevents the lethal effect of colicines on the cultures of microorganisms capable of the biosynthesis of enterocheline.

The capacity of Enterobacteriaceae strains cultivated in a synthetic medium for the biosynthesis of catechols and hydroxamates has been studied. Among the strains under study all strains of the genera Salmonella (8 strains), Escherichia (102 strains), Citrobacter (5 strains), Enterobacter (2 strains), Serratia (1 strain) synthesize catechols. In the genus Shigella (128 strains) all Sh. flexneri serovars (79 strains) do not synthesize catechols, other representatives of this genus synthesize iron-fixing metabolites The synthesis of catecholsin the pathogenic Escherichia serovars is 1.5-2 times lower than in the non-pathogenic and opportunistic enterobacterial strains. Among the representatives of the genus Klebsiella (82 strains) catechols are synthesized by Kl. pneumoniae (68 strains) and not synthesized by kl. rhinoscleromatis (5 strains) and Kl. ozaenae (9 strains). Catechols are not synthesized also by all Proteus organisms under study (12 strains). All the enterobacteria under study show no capacity for the accumulation of hydroxamates under the conditions of catechol synthesis. The intensity of the synthesis of catechols depends on the composition of the medium and the conditions of cultivation.

Pseudomonas Pxy was isolated on p-xylene as sole source of carbon and energy. Substrates that supported growth were toluene, p-methylbenzyl alcohol, p-tolualdehyde, p-toluic acid, and the analogous m-methyl derivatives, including m-xylene. Cell extracts prepared from Pseudomonas Pxy after growth with either p-xylene or m-xylene oxidized the p- and m-isomers of tolualdehyde as well as p-methylbenzyl alcohol. The same cell extracts also catalyzed a "meta" fission of both 3- and 4-methylcatechol. Treatment of Pseudomonas Pxy with N-methyl-N'-nitro-N-nitrosoguanidine led to the isolation of two mutant strains. Pseudomonas Pxy-40, when grown on succinate in the presence of p-xylene, accumulated p-toluic acid in the culture medium. Under the same conditions Pseudomonas Pxy-82 accumulated p-toluic acid and also 4-methylcatechol. When Pseudomonas Pxy-82 was grown on succinate in the presence of m-xylene, 3-methylcatechol and 3-methylsalicylic acid were excreted into the culture medium. A pathway is proposed for the initial reactions utilized by Pseudomonas Pxy to oxidize p- and m-xylene.

Pretreatment with valine (0.5-2.0 mmoles/kg) can suppress the hypothermic response of rats placed in a 4degreesC environment and given d-amphetamine sulfate (5 or 10 mg/kg). The amino acid was most effective when given 30 minutes before amphetamine administration, at which time it also significantly lowered brain tyrosine concentration (and, presumably, suppressed catecholamine synthesis). Because dopaminergic neurons mediate the hypothermic response to amphetamine and because amphetamine's ability to produce hypothermia requires, in part, the release of newly synthesized dopamine, these observed effects of valine pretreatment support the hypothesis that treatments which alter precursor (tyrosine) availability also affect brain catecholamine synthesis.

Enterobacter cloacae strain SS4-56 produced salmonellosis pacifarins when grown in an iron-depleted synthetic medium to which 3.16 X 10-7 g-atom of iron had been added per liter, but did not do so when grown in iron-depleted medium supplemented with 3.16 X 10-4 g-atom of iron per liter. The addition of 3.16 X 10-4 g-atom of ferric iron per liter to a pacifarin-containing culture supernatant fluid had no significant effect upon the ability of the active pacifarins present, when administered per os, to protect mice from an otherwise fatal infection produced by a sequential injection of avirulent and virulent strains of Salmonella typhimurium.

Pseudomonas putida 39/D oxidized p-xylene to cis-3,6-dimethyl-3,5-cyclohexadiene-1,2-diol (cis-p-xylene dihydrodiol). The latter compound was isolated in crystalline form and its physical properties were determined. The cis configuration of the hydroxyl groups in the oxidation product was inferred from its ability to form an isopropylidene derivative with 2,2-dimethoxypropane. Acid treatment of cis-p-xylene dihydrodiol resulted in the formation of 2,5-dimethylphenol. A partially purified preparation of cis-toluene dihydrodiol dehydrogenase oxidized cis-p-xylene dihydrodiol to 1,2-dihydroxy-3,6-dimethylbenzene (3,6-dimethylpyrocatechol). P. putida 39/D oxidized m-xylene to a compound whose spectral and chromatographic characteristics were consistent with the structure 3,5-dimethyl-3,5-cyclohexadiene-1,2-diol. This product was very unstable, and all attempts to isolate it led to the formation of 2,4-dimethylphenol.

The effects of chronic treatment with thyroxine (T4) and estradiol on hepatic microsomal metabolism of estrogens to catechol products were studied and the extent to which activity in vitro correlated with serum estradiol concentrations in vivo was assessed. Female rats were treated with either estradiol benzoate (EB; 56 micrograms/kg/day from silastic implants), T4 (50 micrograms/kg/day, s.c.) or combined EB + T4 for 35 days. Animals treated with EB + T4, but not T4 alone, showed a significant increase above controls both in the concentration of triiodothyronine in serum and food consumption. Serum concentrations of endogenous estradiol in untreated control and T4-treated animals were similar. Although both EB-treated groups received comparable doses of steroid from silastic implants, the concentration of estradiol in serum was 30% lower in EB + T4-treated animals than in animals treated with EB alone. Formation of catechol estrogen metabolites by hepatic microsomes was not significantly altered by EB and T4 administered separately, but enzyme activity was increased significantly with combined hormonal therapy. In contrast, microsomal hydroxylation of testosterone was not increased by treatment with EB + T4, data which suggest that total steroid hydroxylase activity was not enhanced by combined hormonal administration. Correlation analysis of microsomal catechol estrogen formation in vitro with serum concentrations of estradiol in vivo indicated related to the concentration of estrogen in serum only after coadministration of a low dose of T4 with EB.

Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho-substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.

Homogenates of human endometrial, myometrial and leiomyoma tissues were incubated with (2,4,6,7-3H)-estradiol and tritiated catechol estrogens were isolated and identified. Though 2- and 4-hydroxylations were about the same in endometrium, 4-hydroxylation was two to four fold higher than 2-hydroxylation in myometrium and leiomyoma. However, endometrium showed greater capacity to form both 2- and 4-hydroxyestrogens than the other two tissues. Both 2- and 4-hydroxylations were significantly less than in myometrium. In view of the reports indicating that inhibitors of catechol 0-methyl transferase (COMT) might act as antineoplastic agents due to their interference with t-RNA methylases and since catechol estrogens inhibit COMT, the present results suggest that endogenous synthesis of catechol estrogens may play an important role in the pathophysiology of uterine leiomyoma.

A phenol-utilizing yeast, Trichosporon cutaneum POB 14, which has a partially constitutive activity of catechol 1,2-oxygenase, utilized phenol in preference to glucose in a medium containing both phenol (200 mg/liter) and glucose (0.15%) as carbon sources. The glucose consumption was not observed until the concentration of phenol decreased to around 10 mg/liter. This phenomenon was confirmed by [U-14C]glucose uptake experiments. The intracellular activities of hexokinase (EC 2.7.1.1) and catechol 1,2-oxygenase (EC 1.13.1.1) changed inversely when phenol was added during growth in the glucose medium.

Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.

Time course studies of rho-nitroanisole O-demethylation revealed formaldehyde production in excess of rho-nitrophenol (PNP) and 4-nitrocatechol (NTC) formation by rat liver microsomes. This indicated that these products (PNP, NTC) were metabolised further. The hydroxylation reaction PNP yields NTC showed substrate and product inhibition and a requirement for reduced nicotinamide adenine dinucleotide phosphate and O2 and was localized in liver microsomes. It was strongly activated by ascorbic acid, cysteine, adenosine triphosphate or hydroxylamine in vitro and enhanced by phenobarbital treatment in vivo. Mercapturic derivatives were metabolized to the corresponding hydroxy compounds with the same speed as their parent compounds. Both PNP and NTC were metabolized to the corresponding glucuronide and sulfate conjugates. On the other hand, the PNP or NTC glucuronides and sulfates were metabolized with liver microsomes to PNP and NTC.

Although oestriol measurements are well established for the assessment of 'at risk' pregnancies, there are a number of other oestrogens, excreted during pregnancy, which contain additional hydroxyl groups and might be more sensitive indicators of the condition of mother or fetus. Some of these result from the action of hydroxylases possibly present only in the fetus and others from maternal hydroxylations. We review the evidence for the biosynthesis of these polar oestrogens, summarise methods of measurement, and compare values obtained in normal and pathological pregnancies. There is as yet insufficient evidence to enable their potential value to be confirmed.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.

Mutants of Escherichia coli K-12 unable to synthesize the iron-sequestering compound, enterochelin, from 2,3-dihydroxybenzoate have been isolated and divided into three classes on the basis of tests for enzymatic complementation. The genes affected (designated entD, entE, and entF) have been mapped by cotransduction and are located at about minute 14 on the E. coli genome. They were found to be closely linked to other genes (entA, entB, and entC) concerned with enterochelin biosynthesis and a gene (fep) concerned with the uptake of the iron-enterochelin complex. No detectable diffusible intermediate in the formation of enterochelin from 2,3-dihydroxybenzoate was formed by cell extracts of mutants carrying mutations in the entD, entE, or entF genes.

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-(14)C]phenylalanine and l-[U-(14)C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [(14)C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.

The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate --> benzoylformate --> benzaldehyde --> benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the beta-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzymes, benzoylformate decarboxylase and benzaldehyde dehydrogenase, are both induced by benzoylformate. The same benzaldehyde dehydrogenase, or one very similar to it, is also induced by beta-ketoadipate, an intermediate in the subsequent metabolism of benzoate. This dehydrogenase may also be induced by adipate or a metabolite of adipate. These conclusions have been drawn from the physiological and genetic properties of wild-type P. aeruginosa strains and from the study of mutants lacking the second and third enzyme activities.

The metabolism of caffeic acid, previously shown to be carried out by the intestinal microbiota of man and experimental animals, has now been examined in a number of bacteria isolated from human feces. It has been found that of the 12 organisms isolated none has the ability to catalyze more than one reaction of the series leading from caffeic acid to either m-hydroxyphenylpropionic acid or 4-ethylcatechol.

Twenty isolates representing nine bacterial genera were obtained from enrichment cultures and were shown to cometabolize one or more of 22 substituted benzoates. One of the isolates, an Arthrobacter sp., cometabolized m-chlorobenzoate to a product identified as 4-chlorocatechol by thin-layer chromatography and ultraviolet and infrared spectroscopy. The data indicate that cometabolism by the arthrobacter results from the formation of products by its benzoate-oxiding enzyme system that are not acted upon by the catechol-metabolizing enzymes of the bacterium.