Identification of a novel plasmid-associated spectinomycin adenyltransferase gene spd in methicillin-resistant Staphylococcus aureus ST398 isolated from animal and human sources.

OBJECTIVES: Previously described methicillin-resistant Staphylococcus aureus (MRSA) ST398 strains revealed a high frequency of phenotypic resistance to spectinomycin. However, only a few were found to carry the spc resistance determinant. The aim of this study was to identify the genetic mechanism of spectinomycin resistance among spc-negative MRSA ST398 strains. METHODS: Nine spectinomycin-resistant, but spc-negative, MRSA ST398 strains were analysed. The strains were screened for carriage of the spw gene and tested for the presence of transferrable spectinomycin resistance. Plasmid DNA was isolated from all strains and used in transformation assays. The plasmid identified as mediating resistance to spectinomycin was fully sequenced. The function of the novel spectinomycin resistance gene was confirmed by restriction digest inactivation and its distribution was determined using a PCR assay. RESULTS: A single MRSA ST398 strain was spw positive. The remaining strains carried a plasmid that mediated resistance to spectinomycin. Sequence analysis of a single plasmid, termed pDJ91S, revealed that it was 3928 bp in size and contained three open reading frames: a novel spectinomycin resistance gene, designated spd, as well as a repN gene and a rec gene. The XmnI digest inactivation of the spd gene resulted in a 4-fold decrease in spectinomycin MIC. The spd gene was detected in seven other spectinomycin-resistant MRSA ST398 strains that carried a plasmid comparable in size to pDJ91S. CONCLUSIONS: A novel gene, designated spd, that confers resistance to spectinomycin has been identified on a small plasmid in MRSA ST398.

Complete sequence of pSAM7, an IncX4 plasmid carrying a novel blaCTX-M-14b transposition unit isolated from Escherichia coli and Enterobacter cloacae from cattle.

The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.

Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales.

OBJECTIVES: To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. METHODS: UK CTX-M-14-producing E. coli (n=70) from cattle (n=33), turkeys (n=9), sheep (n=2) and humans (n=26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of the bla(CTX-M-14) gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence. RESULTS: The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 isolates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxigenic E. coli ('VTEC'); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of the bla(CTX-M-14) gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n=9), turkeys (n=8) and humans (n=4). All pCT-like plasmids were conjugative, and most were IncK (n=21) and had the same local genetic environment flanking the bla(CTX-M-14) gene (n=23). RFLP analysis demonstrated ≥ 75% similarity among most plasmids (n=22). CONCLUSIONS: pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla(CTX-M-14) genes to different E. coli isolates from humans, cattle and turkeys.

Prevalence of Escherichia coli carrying extended-spectrum ß-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009.

OBJECTIVES: to determine the prevalence of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli from poultry in Great Britain (GB). METHODS: E. coli was isolated from 388 broiler chicken caecal samples from 22 abattoirs and from boot swabs from 442 turkey flocks over successive 1 year periods. CHROMagar ECC with and without cephalosporin antibiotics was used as isolation medium and the chicken study also used CHROMagar CTX. ESBL phenotype isolates were tested for the presence of bla(CTX-M,) bla(OXA), bla(SHV), bla(TEM) and ampC genes(.) CTX-M isolates were tested for O25 serogroup, replicon, CTX-M sequence, multilocus sequence type (MLST), PFGE type, plasmid transfer and qnrA, qnrB, qnrS, qepA and aac(6')-Ib genes. RESULTS: CTX-M-carrying E. coli were isolated from 54.5% of the broiler abattoirs and from 3.6% of individual broiler caecal samples and were CTX-M sequence types 1 (mainly), 3 and 15 with replicon types I1-γ, A/C and P/F, and I1-γ, respectively. CTX-M-carrying E. coli were isolated from 5.2% of turkey meat production farms and 6.9% of turkey breeder farms and were CTX-M sequence types 1, 14 (mainly), 15 and 55 with mainly replicon types F, FIA, K and I1-γ, respectively. None of the CTX-M isolates was serogroup O25. PFGE/MLST showed the CTX-M isolates to be clonally diverse, although MLST 156 with CTX-M-15 was isolated from both chickens and turkeys and has been previously reported in gulls. CTX-M-negative, ESBL- and bla(TEM)-positive strains were mainly TEM-52C. CONCLUSIONS: poultry-derived CTX-M E. coli in GB are different from major CTX-M sequence types causing disease in humans.

Fecal carriage and shedding density of CTX-M extended-spectrum {beta}-lactamase-producing escherichia coli in cattle, chickens, and pigs: implications for environmental contamination and food production.

The number and proportion of CTX-M positive Escherichia coli organisms were determined in feces from cattle, chickens, and pigs in the United Kingdom to provide a better understanding of the risk of the dissemination of extended-spectrum ß-lactamase (ESBL) bacteria to humans from food animal sources. Samples of bovine (n = 35) and swine (n = 20) feces were collected from farms, and chicken cecal contents (n = 32) were collected from abattoirs. There was wide variation in the number of CTX-M-positive E. coli organisms detected; the median (range) CFU/g were 100 (100 × 10(6) to 1 × 10(6)), 5,350 (100 × 10(6) to 3.1 × 10(6)), and 2,800 (100 × 10(5) to 4.7 × 10(5)) for cattle, chickens, and pigs, respectively. The percentages of E. coli isolates that were CTX-M positive also varied widely; median (range) values were 0.013% (0.001 to 1%) for cattle, 0.0197% (0.00001 to 28.18%) for chickens, and 0.121% (0.0002 to 5.88%) for pigs. The proportion of animals designated high-density shedders (≥1 × 10(4) CFU/g) of CTX-M E. coli was 3/35, 15/32, and 8/20 for cattle, chickens, and pigs, respectively. We postulate that high levels of CTX-M E. coli in feces facilitate the dissemination of bla(CTX-M) genes during the rearing of animals for food, and that the absolute numbers of CTX-M bacteria should be given greater consideration in epidemiological studies when assessing the risks of food-borne transmission.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella enterica serovar Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin-α was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols.

AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.

Evaluation of CHROMagar CTX, a novel medium for isolating CTX-M-ESBL-positive Enterobacteriaceae while inhibiting AmpC-producing strains.

OBJECTIVES: The aim of this study was to evaluate CHROMagar CTX (CHROMagar France), a novel agar for the selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene in the presence of enteric bacteria expressing AmpC enzymes. METHODS: A panel of 150 Gram-negative bacteria (mainly Escherichia coli, Enterobacter, Klebsiella, Pseudomonas and Salmonella) isolated from humans and animals were assembled for the purpose of evaluating CHROMagar CTX and comparing it with CHROMagar ECC with the addition of 1, 2, 4 and 8 mg/L cefotaxime or ceftazidime and with bioMérieux extended-spectrum beta-lactamase (ESBL)-Bx agar. CHROMagar CTX was also assessed for its ability to isolate bla(CTX-M) strains from farm animal faeces (n = 342). RESULTS: The panel contained CTX-M-positive (n = 70) strains (CTX-M types 1, 9, 14 and 15), ESBLs (n = 31) belonging to other families (OXA, PER, SHV, TEM, VEB), strains positive for ampC genes (n = 31), strains that overexpressed ampC (n = 6), non-ESBL/AmpC strains (n = 11) and Klebsiella oxytoca (n = 1). CHROMagar CTX was superior to other agars tested for selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene with 100% sensitivity and 64.2% specificity for CTX-M strains in the panel and 90.1% of the colonies from animal faeces plated on CHROMagar CTX were CTX-M strains. CONCLUSIONS: CHROMagar CTX is a valuable agar in situations where it is important to isolate bla(CTX-M) strains in the presence of AmpC strains. The agar may be particularly useful in veterinary studies, where AmpC-producing commensal E. coli can be encountered reasonably frequently in the enteric flora of some animal species and may also be useful, following further evaluation, for samples from humans.

Effect of treatment with 4-hydroxyandrostenedione on the peripheral conversion of androstenedione to estrone and in vitro tumor aromatase activity in postmenopausal women with breast cancer.

The effect of treatment with the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) on the peripheral conversion of androstenedione to estrone has been examined in eight postmenopausal women with advanced breast cancer. Before treatment conversion of androstenedione to estrone ([p]AEIBB) ranged from 0.81 to 3.7% and was almost completely inhibited after treatment with 4-OHA (two doses of 500 mg i.m. with an interval of 12 days between doses). Transfer constants were also measured by the urinary method ([p]AEIBU) for some subjects and decreased from 2.3 +/- 0.52% to 0.24 +/- 0.11% after treatment, a mean reduction of 90%. Mean plasma concentration of estradiol (37.4 +/- 16.6 pmol/liter) and estrone (99.0 +/- 32.2 pmol/liter) decreased significantly (P less than 0.01) to 15.7 +/- 4.6 pmol/liter and 52.4 +/- 8.9 pmol/liter, respectively, after treatment. Aromatase and DNA polymerase alpha (a marker of cell proliferation) activities were measured in seven samples of breast tumor tissue obtained before and after treatment. For three samples there was a marked (67 +/- 17%) decrease in tumor aromatase activity after treatment, for two, little change occurred, while tumor aromatase activity in the other two samples appeared to be resistant to the effect of 4-OHA. The correlation between tumor aromatase and DNA polymerase alpha activities (r = 0.45) failed to reach a significant level.

Longitudinal study of CTX-M ESBL-producing E. coli strains on a UK dairy farm.

The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm.

Hormonal therapies for breast cancer: can progestogens stimulate growth?

The human breast cancer cell line, MCF-7, displays many of the properties of breast tumours in vivo and, in particular, shows similar responses to hormones. Medroxyprogesterone acetate significantly inhibited cell proliferation, but, following continued treatment, this effect was lost and further exposure to the drug stimulated cell proliferation and increased polyamine content while tamoxifen inhibited growth. Prolonged administration of progestogens may thus have adverse effects and should be assessed critically.

Effect of breast cyst fluid on oestrogen 17-oxidoreductase activity in MCF-7 breast cancer cells.

Breast cyst fluid (BCF) was found to stimulate oestrogen 17-oxidoreductase activity in the reductive direction, i.e., conversion of oestrone (E1) to oestradiol (E2), in MCF-7 breast cancer cells. Dialysis of BCF revealed that this property of BCF was present in both dialysed BCF and dialysate, implying that both high and low mol. wt. substances were responsible for stimulating E1 to E2 conversion. Gel filtration of dialysed BCF revealed that the high mol. wt. substances responsible for the stimulation of E1 to E2 conversion had mol. wts. of approximately 11 kD and 68 kD. This property of BCF would serve to increase the concentration of E2, a steroid which may play a role in mammary carcinogenesis.

Steroidal regulation of oestradiol-17 beta dehydrogenase activity of the human breast cancer cell line MCF-7.

We have examined the direct effects of progestins, oestrogens, peptide hormones and growth factors on oestradiol-17 beta dehydrogenase (OE2DH) activity of cultures of the human breast cancer cell line MCF-7. Cells were cultured in the presence of steroid or peptide for 6 days, after which the number of cells was determined and cellular OE2DH activity assessed. Progesterone, 6 alpha-methyl-17 alpha-hydroxyprogesterone acetate, norethisterone and D(-)-norgestrel all profoundly inhibited cell mitosis and stimulated reductive (oestrone----oestradiol-17 beta) and oxidative (oestradiol-17 beta----oestrone) OE2DH activity. Both oestrone and oestradiol-17 beta directly stimulated reductive OE2DH activity, but had no effect on the oxidative direction. Oestradiol-17 beta stimulated cell growth only in phenol-red free culture medium. Ovine prolactin, LH, epidermal growth factor and transforming growth factor did not alter OE2DH activity but small stimulatory effects on the growth of MCF-7 cells were exerted by prolactin and a combination of transforming growth factor with epidermal growth factor. It is concluded that these results may explain, at least in part, the alterations in mitotic activity and tissue oestradiol-17 beta levels observed in breast tissue during varying physiological and pathological conditions, such as during the menstrual cycle and in breast cancers.

Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells.

Gross cystic breast disease is a common benign disease which may be associated with an increased risk for breast cancer. Breast cyst fluid (BCF) contains many steroids, peptide growth factors and proteins. We have now identified interleukin-1 (IL-1) and IL-6 in BCF by specific radioimmunoassays. Concentrations of IL-1 were similar in BCF with low or high Na+/K+ ratios (ratio less than 3 vs greater than 3; 357 +/- 72 pg/ml vs 308 +/- 126 pg/ml). In contrast, IL-6 concentrations were significantly higher (P less than 0.01) in BCF with a Na+/K+ ratio greater than 3 (2.75 +/- 2.34 ng/ml) compared with BCF with a low electrolyte ratio (0.21 +/- 0.09 ng/ml). BCF (10%, v/v) stimulated aromatase activity when added to dexamethasone stimulated breast tumour-derived fibroblasts and there was a significant correlation between the stimulation of aromatase activity and BCF Na+/K+ ratio (r = 0.95, P less than 0.001). A significant correlation was also found between stimulation of aromatase activity and concentration of IL-6 in BCF (r = 0.80, P less than 0.01) but not IL-1 concentration (r = -0.39, not significant). Addition of IL-1 or IL-6 (50 ng/ml) to fibroblasts stimulated aromatase activity but was associated with a small (20%) decrease in cell growth. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast cancer cells.

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Evaluation of a recombinant yeast cell estrogen screening assay.

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment.

The interaction of cytokines in regulating oestradiol 17 beta-hydroxysteroid dehydrogenase activity in MCF-7 cells.

Oestradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) has a pivotal role in the regulation of oestradiol (E2) concentrations in normal and malignant breast tissues. Previous studies have suggested that a number of cytokines can stimulate E2DH activity to increase the conversion of oestrone (E1) to E2. In this investigation we have examined the effect of TNF alpha, interleukin-1 beta (IL-1 beta) and IL-6 on E2DH activity in MCF-7 breast cancer cells. These cytokines may be produced by breast tumours and their presence in conditioned medium (CM) from tumour-derived fibroblasts was also measured to assess their possible contribution to its E2DH stimulatory activity. Treatment of MCF-7 cells with IL-1 beta and TNF alpha (5 ng/ml) significantly increased (P < 0.001) reductive E2DH (red-E2DH, the conversion of E1 to E2) activity. In contrast, IL-6 at a concentration of 100 ng/ml produced little, if any, stimulation of reductive activity. Combinations of all three cytokines acted synergistically to stimulate red-E2DH activity. No cytokine, either alone or in combination, affected oxidative (E2-->E1) activity. Significant concentrations of IL-6 and IL-1 beta were detected in CM, but the stimulation of red-E2DH activity was much greater than that which could be explained by their levels alone. It is concluded that these cytokines may play an important role in regulating E2DH activity in breast cancer cells and may act synergistically in vivo to enhance the formation of E2 in breast tumours.

Regulation of oestradiol 17 beta hydroxysteroid dehydrogenase in breast tissues: the role of growth factors.

Oestradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) is present in normal and malignant breast tissues and regulates the interconversion of oestrone and the biologically active oestrogen, oestradiol. Studies we have previously carried out have indicated that concentrations of oestradiol and the conversion of oestrone to oestradiol are higher in breast tumours than in normal breast tissues. We are currently isolating and characterizing factors produced by breast tumours which are capable of stimulating E2DH (reductive) activity. The production of such factors by breast tumours, which stimulate the conversion of oestrone to oestradiol, would provide a favourable oestrogenic environment to promote tumour growth and may account for the increased concentrations of oestradiol in breast tumours.

Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors.

The aromatase complex has a key role in regulating oestrogen formation in normal and malignant breast tissues. Using dexamethasone-treated fibroblasts, derived from breast tumours, breast tumour cytosol and breast tumour-derived conditioned medium (CM) markedly stimulate aromatase activity. The cytokine, interleukin-6 (IL-6) has been identified as a factor present in CM which is capable of stimulating aromatase activity. To examine whether IL-6 may have a role in vivo in regulating breast tissue aromatase activity, IL-6 production and aromatase activity in breast tumour and adipose tissue from breast quadrants were examined. In 5/6 breasts examined so far, aromatase activity was highest in adipose tissue in the breast quadrant containing the tumour or on which the tumour impinged. There was a significant correlation (P < 0.05, Kendall's rank correlation) between IL-6 production and aromatase activity in these breast tissues. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast tissues.

Biotransformation of genistein in the rat: elucidation of metabolite structure by product ion mass fragmentology.

Biotransformation of the phytoestrogen [14C]genistein was investigated in male and female rats by application of narrow-bore radio-HPLC-MSn (LCQ, Finnigan) to determine intermediates in metabolism. Urine contained five metabolites, Gm1-Gm5, 24 h after dosing by gavage with [14C]genistein (4 mg kg(-1)). Structural analysis following ESI revealed molecular ions [M+H]+ of m/z 447, 449, 273, and 271 for metabolites Gm2, Gm3, Gm5 and genistein, respectively and an [M-H]- of m/z 349 for Gm4. Metabolite structure was deduced by evaluation of product ion spectra derived from unlabelled and [14C]-labelled ions and sensitivity to treatment with beta-glucuronidase. These studies indicated identity of metabolites with genistein glucuronide (Gm2), dihydrogenistein glucuronide (Gm3), genistein sulphate (Gm4) and dihydrogenistein (Gm5). Detection of the beta-glucuronidase resistant major metabolite Gm1 by ESI was poor and so was analysed by negative ion APCI; this revealed a deprotonated molecular ion of m/z 165 which had chromatographic and mass spectral properties consistent with authentic 4-hydroxyphenyl-2-propionic acid, a novel metabolite of genistein. In vitro metabolism studies with anaerobic caecal cultures derived from male and female rats revealed metabolism of genistein to Gm1 via Gm5 and an additional metabolite (Gm6) which was identified from product ion spectra as 6'-hydroxy-O-desmethylangolensin. Biotransformation of genistein by both isolated hepatocytes and precision-cut liver slices was limited to glucuronidation of parent compound. Commonality of genistein metabolites found in rats with those reported in man suggest similar pathways of biotransformation, primarily involving gut micro-flora.