Effect of gonadotrophin-releasing hormone and related analogue on human luteal cell function in vitro

The possible direct effect of gonadotrophin-releasing hormone(GnRH) and GnRH agonist (GnRH-A; buserelin) on basal and humanchorionic gonadotrophin (HCG)-stimulated progesterone (P) andcyclic AMP (cAMP) production by cultured human luteal cellswas examined. Luteal cells from the early or mid-luteal phasewere incubated in long-term cultures. They responded to HCGstimulation with a 2- to 3-fold increase in P production anda 2-fold increase in cAMP production. The addition of GnRH (10^–7and 10^–5 M) or GnRH-A (10^–7 and 10^–5 M) tothe medium had no effect on either basal or HCG-stimulated secretion.These results indicate that both GnRH and GnRH-A have no directeffect on human luteal steroidogenesis in vitro.     

Bradykinin induces contractions of the human ovarian follicular wall in vitro

Bradykinin has been shown to have a positive modulatory rolein the ovulatory process. In the present study, the contractileproperties elicited by bradykinin (10^–l2–10^–5M) in the isolated human follicular wall were examined. Stripsfrom the follicular base and apex were mounted separately andsuperfused at 37°C with oxygenated HEPES buffer in tissuechambers. The contractile activity was recorded isometricallyby a force transducer (Grass model FT03), under a passive tensionof 5 mN. Preovulatory follicles (cycle day 10–14) weremore sensitive to bradykinin than follicles of an earlier developmentalstage (cycle day 1–9). The response to bradykinin wasnot altered by the addition of indomethacin (10^–7 M),atropine (10^–6 M) or phenoxybenzamine(10^–7 M). Thesedata suggest that the contraction caused by bradykinin is aspecific effect, which, under physiological conditions, mightcontribute to the ovulatory process by inducing a rise in tonein the follicle thereby facilitating the extrusion of the oocytethrough the digested follicular apex.     

Effect of clomiphene isomers on progestin synthesis in cultured human granulosa cells

The effect of en-clomiphene and zu-clomiphene (10^–9–10^–5M) on progestin synthesis in cultured human granulosa cellswas studied under basal conditions and in the presence of LH(100 ng/ml). Granulosa cells were obtained from either pre-ovulatoryfollides of clomiphene-HMG-stimulated cycles or from large folliclesof mid-to-late follicular phase of spontaneous cycles. The basaland LH-stimulated progesterone accumulation was dose-dependentlyreduced by en- and zu-clomiphene (10^–6–10^–5M) in cells from both groups studied, an effect similar to thatof oestradiol. In contrast to oestradiol, both en- and zu-clomiphene(10^–6–10^–5 M) reduced the basal and LH-stimulatedpregnenolone accumulation in cells of stimulated cycles. Theeffect of clomiphene on progesterone, and pregnenolone accumulationwas more pronounced in LH-stimulated cells than under basalconditions. There were no qualitative differences between thetwo isomers and the results were principally the same in cellsfrom both groups of follicles studied. It is concluded thaten-clomiphene and zu-clomiphene have similar inhibitory effectson progestin synthesis in human granulosa cells in vitro.     

Endometriosis: Secretion of interleukin-6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids

Endometriosis is generally associated with an immuno-inflammatoryprocess that takes place in the peritoneal cavity of patients.Interleukin (IL)-6, a multifunctional cytokine involved in numerousimmunological and prolifer-ative processes, has been found athigh concentrations in the peritoneal fluid of endometriosispatients. The purpose of this study was to investigate the abilityof endometriotic cells to produce IL-6 and to assess the regulationof its secretion by proinflammatory cytokines and sex steroids.Cultures of human endometriotic cells were exposed to differentconcentrations of cytokines and sex steroid hormones for varyingperiods of time. IL-6 secretion was measured using an enzyme-linkedimmunosorbent assay. Endometriotic cells spontaneously releasedIL-6 in culture. IL-1 and tumour necrosis factor (TNF)- (0.1–100.0ng/ ml) potentiated IL-6 secretion in a time- and dose-dependentmanner. Interferon- (0.4–400 ng/ml) induced a dose-relatedincrease in IL-6 secretion and showed a synergjstic effect onthat secretion in combination with TNF- (10 ng/ ml). Eitherspontaneous or cytokine-induced IL-6 secretion was inhibitedby progesterone (10^–8–10^–5 M) and danazol(10^–6 M), whereas oestradiol (10^–8–10^–5M) had a limited inhibitory effect. The antiprogestin RU486(l0^–8–10^–4 M) antagonized the inhibitory effectsof progesterone and danazol, but showed agonist action whenused alone. These findings indicate that endometriotic tissuemay actively contribute to the biological changes observed inthe peritoneal fluid of endometriosis patients. They also providenew insights into the mechanisms of action of progesterone andthose of danazol and RU486 used in the treatment of endometriosis.     

Pregnancy: Activation of protein kinase C is required for oxytocin-induced contractility in human pregnant myometrium

Intracellular mediators regulating the initiation of parturitionare not fully understood. This study was designed to determinethe possible mechanism of oxytocin-induced uterine contractilityduring labour. In-vitro isometric contraction studies were performedwith longitudinal strips of human pregnant myometrium in thepresence and absence of the protein kinase C inhibitors, staurosporineand RO 31–8220, and the tyrosine kinase inhibitor, genistein.Phospholipase D activity was measured by employing the transphosphatidylationreaction. Staurosporine significantly reduced oxytocin-stimulatedcontractile activity with mean activity reduced by >50% followingthe addition of 10^–6M staurosporine (P < 0.01), whileaddition of 10^–5 M resulted in a measured mean contractileactivity of –10% of the control (P < 0.001, n = 5).Similarly, uterine activity was minimal with oxytocin applicationfollowing incubation with RO 31–8220, mean contractileactivity being reduced by -40% by the addition of 10^–7M RO 31–8220 (P < 0.05) and by –87% by the additionof either 10^–6 or 10^–5 M (P < 0.01, n = 3). Conversely,addition of genistein (10^–7 and 10^–6 M) had littleeffect on oxytocin-induced contractions, although at a higherconcentration (10^–5 M) a significant reduction in oxytocin-inducedcontractile activity was observed (P < 0.01). Oxytocin evokedphospholipase D activation in a concentration- and time-dependentmanner in cultured human pregnant myometrial cells (n = 4).These results indicate that activation of protein kinase C andtyrosine kinase are involved in the regulation of oxytocin-mediatedmyometrial contractile activity and that a coupled phospholipaseD/phosphatidate phosphohydrolase pathway may play a role inthe sustained stimulation of myometrial activity during labour.     

Predominant role for nitric oxide in the relaxation induced by acetylcholine in human uterine artery

The effect of acetylcholine on the isolated human uterine arteryrings was investigated. Acetylcholine (10^–10 M to 6x 10^–5M) induced concentration- and endothelium-dependent relaxation(pD2=7.4±0.02, maximal response was 77.5±6.3%of relaxation induced by papaverine at 3x10–4 M), diethylcarbamazine(104 M) and tetra-ethylammonium (3x10^–4 M) had no effectson acetylcholine-evoked relaxation. Methylene blue (10–5M) and NG-monomethyl-L-arginine (L-NMMA) (3x10^–6 to 3x10^–5 M) antagonized relaxation induced by acetylcholine.The inhibition of endothelium-dependent relaxation by L-NMMA(10–5 M) was reversed by L-arginine (10–5 M) butnot by D-arginine (10^–4 M). It is concluded that in uterineartery acetylcholine induces endothelium-dependent relaxation.It is suggested that the acetylcholine-induced relaxation ofisolated uterine artery is probably mediated via endothelialnitric oxide formation     

Arachidonic acid and its lipoxygenase metabolites stimulate prolactin release in superfused pituitary cells

The direct effect of leukotrienes and other lipoxygenase productson prolactin release has been assessed. Arachidonic acid andits lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoicacid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetraenoic acid(15-HETE) stimulated the release of prolactin in superlusedrat pituitary cells in a dose-dependent manner. Leukotrienes(LT) A₄, B₄, C₄ and E₄ provoked a very marked biphasic and dose-dependentsecretion of prolactin from superfused cells. Maximal effectswere achieved with leukotrienes at a concentration of 3 x 10^–11to 3 x 10^–10 M but LTD₄ did not affect peptide releaseunder these conditions. The metabolites were more potent thanarachidonic acid in affecting hormone secretion. Pukes of 4mioutes duration of these fatty acids may even elicit a morepronounced response thao thyrotrophin-releasing hormone (TRH).Nordihydroguaiaretic acid (NDGA 10^10–6 M), a lipoxygenaseinhibitor, prevented the effect of arachidonic acid on peptidesecretion. Repeated TRH (10^–7 M) administration to pituitarycells led to a reduction in cell response, which may also beobserved in cells pre-treated with pulsatile 5-HETE or 15-HETE.These data support previous findings that arachidonic acid andits lipoxygenase metabolites may play a role in the secretorymechanism of prolactin release in pituitary cells.     

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.     

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis

The objective of this study was to investigate the influenceof thyroid hormone on gonadotrophin-induced oestradiol and progesteronesecretion by human granulosa cells maintained in vitro. Granulosacells were obtained by aspiration of pre-ovulatory folliclesfrom women undergoing assisted reproductive technology. Ovulationinduction was performed with gonadotrophin-releasing hormoneagonist, human menopausal gonadotrophin and human chorionicgonadotrophin. Granulosa cells were maintained in vitro in adefined medium with added insulin. Between 48 and 72 h afterthe initiation of cell culture, oestradiol and progesteronesecretion into the medium was determined for granulosa cellsgrowing in serum-free medium with follicle-stimulating hormone(FSH)/luteinizing hormone (LH) and in serum-free medium withFSH/LH and thyroxine added in a concentration range of 10^–10–10^–7M. All concentrations of thyroxine used produced a statisticallysignificant increase in oestradiol (range 1.18–1.37 timesthe amount with FSH/LH alone) and progesterone (range 1.29–1.51times the amount with FSH/LH alone) secretion.     

Sandostatin has no direct effect on ovarian steroidogenesis in vitro

Rat granulosa and theca—interstitial cells from immature,oestradiol-treated rats were isolated and incubated for 144h with follicle stimulating hormone (FSH), luteinizing hormone(LH), insulin alone or in combinations, and with two doses ofsandostatin (10^–7 M and 10^–6 M per culture). Oestradioland testosterone production by granulosa and theca—interstitialcells, respectively, was measured in culture media. The stimulatoryeffects of FSH alone and FSH with insulin but not insulin aloneon oestradiol production by granulosa cells were observed. Similarly,increased testosterone concentrations after treatment with LHalone and LH with insulin but not insulin alone were found inmedia from theca—interstitial cells. The addition of highor low doses of sandostatin to the cultures did not affect theproduction of oestradiol or testosterone. It was concluded thatsandostatin does not exert any direct effect on ovarian steroidogenesisin vitro.     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17 (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.     

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge

DNA vaccination offers the advantages of viral gene expressionwithin host cells without the risks of infectious virus. Likeviral vaccines, DNA vaccines encoding internal influenza virusproteins can induce immunity to conserved epitopes and so maydefend the host against a broad range of viral variants. CD8⁺cytotoxic T lymphocytes (CTL) have been described as essentialeffectors in protection by influenza nucleoprotein (NP), althougha lesser role of CD4⁺ cells has been reported. We immunizedmice with plasmids encoding influenza virus NP and matrix (M).NP + M DNA allowed B6 mice to survive otherwise lethal challengeinfection, but did not protect B6-ß₂m(–/–)mice defective in CD8⁺ CTL. However, this does not prove CTLare required, because ß₂m(–/–) mice have multipleimmune abnormalities. We used acute T cell depletion in vivoto identify effectors critical for defense against challengeinfection. Since lung lymphocytes are relevant to virus clearance,surface phenotypes and cytolytic activity of lung lymphocyteswere analyzed in depleted animals, along with lethal challengestudies. Depletion of either CD4⁺ or CD8⁺ T cells in NP + MDNA-immunized BALB/c mice during the challenge period did notsignificantly decrease survival, while simultaneous depletionof CD4⁺ and CD8⁺ cells or depletion of all CD90⁺ cells completelyabrogated survival. We conclude that T cell immunity inducedby NP + M DNA vaccination is responsible for immune defense,but CD8⁺ T cells are not essential in the active response tothis vaccination. Either CD4⁺ or CD8⁺ T cells can promote survivaland recovery in the absence of the other subset.     

Endocrinology and Paracrinology: Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells

Cyclic AMP (c-AMP)-dependent cell shape changes can be usedto study the gonadotrophin response in cultured human granulosa-luteincells. The same approach has been developed here to determinethe direct potential antigonadotrophic effect of gonadotrophin-releasinghormone (GnRH) and a GnRH agonist (triptorelin) on human granulosa-luteincells. Treatment with triptorelin or GnRH alone for 1 h didnot affect granulosa-lutein cell morphology. However, in thepresence of stimulatory doses of follicle stimulating hormone(FSH), triptorelin 5x10^–7–5x10^–6 M) and GnRH(10^–11–10^–9 M) inhibited the FSH-induced c-AMP-dependentresponse. The antigonadotrophic effect of triptorelin was preventedby two GnRH antagonists, indicating that triptorelin acts viaspecific GnRH binding sites. On the other hand, triptorelinfailed to inhibit human chorionic gonadotrophin- and forskolin-mediatedmorphological changes. Our results suggest that the GnRH agonistinteracts specifically with the FSH-induced c-AMP-dependentcascade of events, at a site located ahead of that of c-AMPgeneration. In conclusion, GnRH and triptorelin strongly inhibitFSH-mediated function in human granulosa-lutein cells in culture.This inhibition might play a role in the low follicular developmentrates observed in some patients treated with GnRH agonist +gonadotrophins for ovarian stimulation. cell shape changes/FSH/GnRH/human granulosa-lutein cells/triptorelin     

Exogenous steroids and the control of oestradiol secretion by human granulosa-lutein cells by follicle stimulating hormone and insulin-like growth factor-I

This study first examined the relative activities of 17-hydroxylase,17, 20-lyase and aromatase in human granulosa–lutein cellsby challenging the cells with steroid precursors in the oestradiolbiosynthetic pathway. When cells from four patients were challengedwith precursor steroids on the pathway to oestrogen synthesis(pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesteroneand androstenedione at 5 x 10^–6 M), oestradiol (nmol/l)outputs after 1 day of culture were (median, interquartile range)as follows: 4.1 (2.1– 8.8; pregnenolone), 3.1 (1.7–6.0;progesterone), 12.5 (6.9–18.1; 17hydroxypregnenolone),8.2 (4.1–16.7; 17hydroxyprogesterone) and 251 (140–819;androstenedione). No further increases were seen when the steroidconcentration was increased to 1 x 10^–5 M. Basal oestradiolsecretion was 3.5 (1.6–8.2) nmol/l. We conclude that theconversion of pregnenolone/progesterone to oestradiol by granulosa–luteincells is rate limited by 17-hydroxylase activity but that thesecells are capable of oestradiol secretion (in the nmol/l range)in the absence of androstenedione. In the second part of thisstudy we examined the control of granulosa–lutein oestradiolsecretion by follicle stimulating hormone (FSH) and insulinlikegrowth factor-I (IGF-I) in the presence and absence of exogenousandrostenedione (10^–6 M). Cells were cultured for up to6 days and basal oestradiol (nmol/l) fell dramatically overthis period both in the presence and absence of androstenedione,e.g. from 339 (223–419) (median and interquartile range,cells from five patients cultured in the presence of androstenedione)after 2 days to 14 (7–59) after 6 days. There was no effectof FSH (83/575, 0–160 IU/l) in the absence of androstenedionebut in its presence FSH (96 IU/l) increased oestradiol secretionslightly by 153 ±45 (day 4) and 151 ± 21 (day6; results mean ± SEM, percentage increase over time-matchedcontrols; cells from five patients). In contrast, the effectof IGF-I (30 ng/ml) was to markedly enhance oestradiol secretionto 1099±320% (n = 7) of the control (day 4) in the absenceof exogenous androgen and to 551 ± 184% (n = 5) in itspresence. There was no evidence of any synergistic interactionbetween IGF-I and FSH during the culture period in the absenceof androstenedione. However, there was a synergistic effectfor IGF-I (30 ng/ml)/FSH (96 IU/l) after 6 days in culture inits presence in that oestradiol secretion increased by 1748± 294% (n = 5) compared to 157 ± 21% (FSH) and1211 ± 233% (IGF-I) for the stimulators on their own.However, this effect may be explained, in part, by the increasein cell number provoked by IGF-I over this culture period. Weconclude that (i) under these conditions granulosa cells showedlow 17-hydroxylase activities, (ii) granulosa cells are capableof synthesizing oestradiol in the absence of exogenous androgens,the substrates for the aromatase complex, and (iii) FSH is oflittle importance in stimulating oestradiol secretion from granulosa-luteincells, and the evidence for it positively modulating IGF-I activityis poor.     

Endocrinology: Somatostatin in physiological concentrations inhibits basal and enhances luteinizing hormone-stimulated progesterone release from human granulosa--luteal cells

To study the effect of somatostatin on ovarian function, weinvestigated the action of physiological concentrations of somatostatin(5.0x10–¹²–1.0x10–¹⁰ M) on the basal and luteinizinghormone (LH)-stimulated progesterone release from cultured humangranulosa—luteal cells obtained from in-vitro fertilizationpatients. Somatostatin exerted a significant and inhibitoryeffect on basal progesterone release from the granulosa—lutealcells, whereas it was unable to inhibit LH-stimulated progesteronerelease. Instead, a significant increase in progesterone releasewas observed after concomitant incubation with LH and somatostatincompared with the untreated controls. We suggest that somatostatinmay serve as a regulator of ovarian functions under physiologicalconditions.     

Action of leukotriene B4 in human granulosa cells in vitro

Leukotriene B4 directly enhanced progesterone release from superfusedhuman granulosa cells. This secretory effect was observed inconcentrations from 10^–12 to 10^–10 M. Lower andhigher concentrations failed to affect progesterone release.When we analysed the high performance liquid chromatographyprofile of supernatant from human granulosa cell cultures, wedetected a leukotriene B4 peak. In conclusion, these data supportthe hypothesis that leukotriene B4 may participate in the intracellularmechanism of progesterone release in human granulosa cells.     

Regulators of sperm function: Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa

The involvement of protein kinases in platelet activating factor(PAF)-induced acrosome reaction of human spermatozoa was investigatedusing specific inhibitors of protein kinase A (PKA), proteinkinase C (PKC) and protein tyrosine kinase (PTK). PAF (10^–9–10^–11M) treatment of spermatozoa enhanced the acrosome reaction ina dose-dependent manner (32 ± 4% at 10^–9 M, 28±4%at 10^–10 M and 24 ± 3% at 10^–11 M respectively).When spermatozoa were preincubated with PKA, PKC and PTK inhibitor(KT5720, calphostin C and genistein) for 15 min prior to additionof PAF, there was a significantly reduced acrosome reactioninduced by PAF, but complete inhibition was not observed. Onthe other hand, combined use of three inhibitors completelyinhibited PAF-induced acrosome reaction to levels of non-treatedsamples. These results suggest that the induction of the acrosomereaction by PAF treatment may involve the activation of PKA,PKC and PTK signalling pathways, and that interaction betweenthese pathways may regulate complex mechanisms of PAF-inducedacrosome reaction. acrosome reaction/human spermatozoa/platelet activating factor/protein kinase     

Aneuploidy in male Indian muntjac cells is limited to the Y2 chromosome

In untreated cell cultures of Indian muntjac (Munticus muntiacusvaginalis; 2n = 6, 7) we observed that spontaneous aneuploidyis limited primarily to the Y₂ chromosome. We therefore treatedthe cells with aneugenic agents to determine if induced aneuploidyfollows the same pattern and, hence, if there are limitationson the generation of aneuploidy or survival of cells lackingcertain chromosomes. Exposing the cells to benomyl (8–100µg/ml for 1 h), caffeine (5 x 10^–5–2x10^–4for 2, 24 and 72 h) and colchicine (2 x 10^–4 and 5 x 10^–5M for 1, 24, 48 and 72 h) resulted in cells primarily aneuploidfor the Y₂ chromosome. The frequency of cells lacking Y₂ wasfar higher than those having an extra Y₂ chromosome. The frequencyof cells aneuploid for all other chromosomes combined was muchlower than that for Y₂. The data imply that the Indian muntjacgenome can tolerate loss of the Y₂ chromosome only and thataneuploidy for other chromosomes might cause lethality. Thismight be because the small number of chromosomes in the genomepredisposes aneuploid cells to an imbalance of genes carryingout the basic activities required for cell division and cellsurvival. Because of the small chromosome number, the largesize of the chromosomes and the ease of distinguishing everychromosome without banding or any other special treatment, e.g.FISH, this system could be useful and convenient in the studyof induction of aneuploidy. This simple and inexpensive systemcan be utilized as a screening system for preliminary studiesdealing with induced aneuploidy. ¹To whom correspondence should be addressed. Tel: +1 702 784 6544; Fax: +1 702 784 1302; Email: vig{at}med.unr.edu     

Endocrinology: Prostaglandin F2{alpha} stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells

Human ovarian follicular fluid contains a number of insulin-likegrowth factor binding proteins (IGFBP) of which IGFBP-3 is themost abundant. IGFBP-3 synthesis is growth hormone-regulated.We studied the effect of prostaglandin F₂ (PGF₂) on IGFBP-3secretion by cultured human granulosa-luteal cells from follicularaspirates of women participating in an in-vitro fertilizationprogramme. The IGFBP-3 concentration was measured using a specificmonoclonal immunofluorimetric assay. Contrary to a previousreport on unstimulated follicles, this study demonstrated apositive correlation between follicular fluid IGFBP-3 concentrationand follicular size. PGF2a was found to stimulate in a dose-dependentfashion the secretion of IGFBP-3. Significant (p < 0.05)effects were found at PGF₂ concentrations of 10^–8, 10^–7and 10^–6 M. Because IGFBP-3 inhibits progesterone productionstimulated by insulin-like growth factor (IGF)-I, the PGF₂-inducedstimulation of IGFBP-3 production may be one of the mechanismswhereby PGF₂ exerts its luteolytic effect via the IGF system     

A correlation of Salmonella mutagenicity with DNA adducts induced by the cooked-food mutagen 2-amino-1-methyl-6-6-phenylimidazo[4,5,-b]pyridine

The correlation of bacterial mutagenicity with DNA adducts fromthe heterocyclic amine cooked-food mutagen 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) was investigated in Salmonella typhimurium strains TA98(uvrB deficient) and TA1978 (uvrB proficient). Bacterial cellswere exposed to PhIP using a modification of the Ames/ Salmonellamicrosuspension assay. Half of the cells, generated from a 90min pre-incubation and washing, were plated for revertant formationwhile the remaining half was subjected to DNA adduct analysisvia ³²P-postlabeling. In TA98, DNA adducts were detected atan RAL (relative adduct labeling) of 10x10^–7 and 21x10^–7at PhIP concentrations of 5.5 and 17 µM, respectively.This corresponded to 28.8 and 20.9 adducts/revertant, respectively.These values were based on the assumption that only four repeatingGC bases within a 75 DNA base region is the gene target sitefor PhIP induced mutations. In TA1978, no revertants above backgroundwere detected at any concentration of PhIP tested. DNA adducts,however, were detected at 11x10^–7 and 21x10^–7 adductsper nucleotide at 223 and 1116 µM PhIP, respectively.The lack of detectable revertants, but the presence of DNA adducts,suggests pre-mutational lesions did occur during the 90 minpre-incubation. Presumably, when the S9 activating system andPhIP were removed (via washing with phosphate buffered saline)prior to plating, the cells containing an intact uvrB repairsystem repaired the lesions during the incubation time on theplates. In conclusion, the induction of revertants by adductsappears quite efficient, as