NSAID-induced apoptosis in Rous sarcoma virus-transformed chicken embryo fibroblasts is dependent on v-src and c-myc and is inhibited by bcl-2.

Mounting epidemiological and experimental evidence implicates non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

In vivo and in vitro expression of a non-mammalian cyclooxygenase-1.

Unlike cyclooxygenase 2 (COX-2), COX-1 has never been identified, purified or cloned in a non-mammalian species. Here we report the RT-PCR cloning of a chicken cDNA that encodes the amphipathic membrane binding region and parts of the dimerization and catalytic domains of COX-1-like enzyme. Sequence comparison showed this putative COX-1 to be evolutionarily less conserved than COX-2. Furthermore, whereas COX-1 in mammals is broadly expressed in tissues as a constitutive enzyme, the mRNA detected by our clone in chicken was almost absent in tissues and embryo fibroblasts (CEF). Highest expression was in brain and seminal vesicle. This transcript was not detectable during chick embryogenesis and, as is the case for mammalian COX-1, was not induced above background by mitogen stimulation. The identification of an avian COX-1 shows that COX-1 and COX-2 existed as separate catalysts for prostaglandin synthesis before the divergence of birds and mammals.

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Drug inhibition and cellular regulation of prostaglandin G/H synthase isoenzyme 2.

Prostaglandin G/H synthase isoenzyme 2 (PGHS-2) was identified as an immediate-early gene product induced by Rous sarcoma virus, serum, phorbol ester and a wide variety of other mitogens. Induction of PGHS-2 occurs through an increase in PGHS-2 gene transcription. Dexamethasone inhibits both the basal and induced levels of PGHS-2 mRNA. In contrast, PGHS-1 gene transcription rate, mRNA, and protein levels are unaffected by mitogens and dexamethasone. Post-transcriptional down-regulation of PGHS-2 mRNA plays a significant role in causing dexamethasone's effect. Specific cell systems have been identified which allow selective analysis of PGHS-1 and PGHS-2 at the nucleic acid and protein levels. These cell systems indicate that PGHS-1 and PGHS-2 may have different sensitivities to non-steroidal anti-inflammatory drugs. Furthermore, inhibition of PGHS activity correlates well with suppression of the transformed phenotype in an in vitro cell model.

Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.

We have previously reported the isolation and characterization of a new form (PGHS-2) of prostaglandin G/H synthase (PGHS, cyclooxygenase) from chicken embryo fibroblasts. To further study the regulation and structure of the gene, we have cloned the entire chicken PGHS-2 (previously termed miPGHSch) gene with its 5'-flanking region from a chicken genomic library. A genomic Southern blot showed the existence of a single PGHS-2 gene. The size of the gene was estimated at 8.9 kb through DNA sequencing and polymerase chain reaction analysis. The PGHS-2 gene was found to contain 10 exons, giving it a structure similar to that of the human PGHS-1 and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6 kb of the 5'-flanking region immediately upstream of the transcription start site was determined. The promoter sequence contained a TATA box and a variety of enhancer elements, including a serum response element, an AP-1, an NF-kappa B, and several SP-1 and AP-2 sites. Chloramphenicol acetyltransferase (CAT) assays showed that the first 158 nucleotides of the promoter efficiently drove transcription of the CAT reporter gene in serum-stimulated cells. Dexamethasone, a potent inhibitor of prostaglandin synthesis, had no effect on CAT activity, although this drug is known to markedly decrease PGHS-2 mRNA in vivo. This suggests that dexamethasone may inhibit PGHS-2 mRNA expression at the post-transcriptional level. Analysis of hamster/mouse somatic cell hybrids with radiolabeled cDNA probes demonstrated that PGHS-1 mapped to chromosome 2 and PGHS-2 mapped to chromosome 1 of the mouse genome.

An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program.

OBJECTIVE: To select in vitro fertilization (IVF) patients with a low sperm penetration assay (SPA) value and to determine if the penetration rate, fertilization rate, and the pregnancy rate (PR) can be improved in these patients by treating sperm with refrigeration, calcium ionophore A23187, prostaglandin E1, prostaglandin E2, or heparin. DESIGN: The study consists of three parts: identification of patients with poor SPA values, analysis of treatments to improve the SPA value, and evaluation of the treatments to improve fertilization and PRs. RESULTS: The data indicate that treatment of sperm with refrigeration can improve fertilization and PRs during IVF in selected patients previously shown to have an improved SPA value with the treatment. CONCLUSIONS: Three points are emphasized: (1) the treatments analyzed in this study can improve SPA values in some of the patients with low sperm penetration capacity; (2) of the treatments studied, sperm refrigeration resulted in the largest improvement in sperm penetration capacity; and (3) sperm refrigeration can increase fertilization and PRs during IVF in this select group of patients.

Diethylstilbestrol-induced perinatal lethality in the rat. I. Relationship to reduced maternal weight gain.

An analysis was performed to determine the mechanism of depressed maternal weight gain and its effect on perinatal lethality following prenatal exposure to diethylstilbestrol (DES). Pregnant Sprague-Dawley rats were dosed orally by gavage with DES or corn oil (control) during various intervals of gestation. The maternal weight-gain patterns of control and treated dams and the number of live offspring were recorded. The amounts of feed and water intake and feces and urine output in pregnant dams were measured, and metabolic rate and thyroid hormone levels were also determined. DES (at 45 micrograms/kg/day) was embryo- and fetolethal during implantation and parturition, and there was an accompanying decline in maternal weight. Growth of adult males, nonpregnant females, and weanlings of both sexes was also depressed. During pregnancy, the net intake of feed and water was not altered by the drug, but maternal serum thyroxine and metabolic rate were significantly elevated. Reduced metabolic efficiency, then, is the likely mechanism for weight depression. Reduction of maternal weight gain during pregnancy by DES is a diagnostic indicator of fetolethality, but is probably not causally related to it.

Diethylstilbestrol-induced perinatal lethality in the rat. II. Perturbation of parturition.

Evidence is presented suggesting that the fetolethal properties of diethylstilbestrol (DES) are indirect, mediated maternally through a perturbation of the normal mechanisms of parturition. Oral administration of the compound to Sprague-Dawley rats near Day 18 of pregnancy was shown to delay the onset of parturition, prolong labor, and induce dystocia, with a concomitant large increase in perinatal mortality. Exposure during Days 8-16 was without effect, whereas treatment in the Day 18-20 interval resulted in preterm delivery. Inability to initiate labor at term, accompanied by fetal death, also resulted from the administration of hCG on Days 16-18. The relative incidence of stillbirths in DES-exposed pups was markedly decreased by Caesarean delivery. The average weight of the maternal pituitary gland was not affected by treatment, whereas maternal adrenal glands were 30% larger. Maternal blood levels of corticosterone were not significantly elevated, however. The average number of follicles on Day 21 was significantly reduced by DES, and a histological analysis failed to demonstrate a luteotropic effect of the compound. In dams treated on Days 8-18, serum progesterone was reduced by as much as 60%, and total estrogens were 32% lower than in controls. We conclude that DES acts in the rat to depress the preterm levels of steroid hormones, which leads to a failure of uterine contraction accompanied by placental detachment and fetal death.

Perinatal toxicity of ethylene glycol dimethyl ether in the rat.

Ethylene glycol dimethyl ether (EGdiME) was administered by gavage to pregnant Sprague-Dawley rats in doses of 30, 60, 120, 250, 500, and 1000 mg/kg/day from day 8 through day 18 of gestation. The effects of the compound on maternal weight gain, length of gestation, perinatal mortality, teratogenicity, average fetal weight on day 19, and average pup weight one day after birth were assessed. A clear pattern of dose-dependent maternal and fetal toxicity was observed. EGdiME caused maternal deaths at 1000 mg/kg/day and was fetolethal at doses ranging from 120 to 1000 mg/kg/day. A dose of 60 mg/kg/day resulted in a 7% weight decrease and severe edema in pups surviving to birth. Skeletal examinations in this group revealed fetotoxicity as evidenced by the lack of ossified bone, but there was no indication of anomalies in soft tissues. The same concentration in dams allowed to go to term resulted in a delay in the onset of parturition and produced litters with only one-third the number of live pups as controls. Of these, an average of less than 1 per litter survived to day 1 postpartum. The compound was not fetolethal on day 19 at a dose level of 30 mg/kg/day. Perinatal mortality in the interval between day 19 of gestation and birth was manifested, however, by an average reduction of 2 live pups per litter at birth. There was a close correlation between the fetotoxic effects of the various concentrations and the degree to which the maternal weight gain pattern of each departed from the control profile.

Fetotoxic alterations in the normal ontogenies of rat microsomal and lysosomal enzymes.

The activity patterns during development for acid phosphatase (Ac-P), alkaline phosphatase (A1-P), beta-glucuronidase (beta G), and UDP-glucuronyltransferase (UDPGT) have been determined in various tissues of the rat for corn oil and distilled water controls as well as in animals prenatally exposed to four fetotoxic chemicals. Postnatal assays were performed on both sexes separately. In control animals, tissue-specific differences between male and female activity levels were found for UDPGT. In the liver of mature offspring, enzyme activity was greater in males than in females. Although no sex difference was observed in the intestine, the kidneys of females exhibited higher values than those of males. An original computer-assisted methodology is presented, designed (a) to permit a mathematical description for the complex curves exhibited by these ontogeny profiles, and (b) to assess the statistical significance of chemical-induced alterations in these complex developmental patterns, specifically, to target sensitive periods and subtle changes near the fetotoxic threshold. Oral administration (days 6-18 of gestation) of 3,3',4,4'-tetrachlorobiphenyl (4CB) to pregnant females resulted in an induction of liver UDPGT activity in offspring postnatally, and some alterations in the perinatal pattern of beta G in the same tissue. This treatment also produced differences in the intestinal patterns of Ac-P and male UDPGT. No significant changes were observed in offspring exposed to diethylstilbestrol (DES). Treatment with zeranol (ZN) caused reductions in activity over the entire postnatal period for beta G in liver, brain, intestine, and kidney, for A1-P in brain, and for Ac-P in the intestine. Cadmium-treated dams gave birth to offspring that exhibited slightly altered ontogenies only in intestine for UDPGT and AcP. The alterations in these developmental profiles indicate periods of increased sensitivity, and may be useful in directing more specific studies into the fetotoxic mechanisms of these compounds.

Effect of maternal exercise on fetal liver glycogen late in gestation in the rat.

To determine the effect of maternal exercise on fetal liver glycogen content, fed and fasted rats that were pregnant for 20.5 or 21.5 days were run on a rodent treadmill for 60 min at 12 m/min with a 0% grade or 16 m/min up a 10% grade. The rats were anesthetized by intravenous injection of pentobarbital sodium, and fetal and maternal liver and plasma samples were collected and frozen. Fetal liver glycogenolysis did not occur as a result of maternal exercise. Fetal blood levels of lactate increased 22-60%, but glucose, plasma glucagon, and insulin were unchanged during maternal exercise. Maternal liver glycogen decreased as a result of exercise in all groups of rats except the fasted 20.5-day-pregnant group. Plasma free fatty acids increased in all groups and blood lactate increased in fed (20.5 days) and fasted (21.5 days) pregnant rats. Maternal glucose, glucagon, and insulin values remained constant during exercise. The fetus appears to be well-protected from metabolic stress during moderate-intensity maternal exercise.

Developmental toxicity of nine selected compounds following prenatal exposure in the mouse: naphthalene, p-nitrophenol, sodium selenite, dimethyl phthalate, ethylenethiourea, and four glycol ether derivatives.

Ethylene glycol dimethyl ether (EGdiME), diethylene glycol dimethyl ether (diEGdiME), triethylene glycol dimethyl ether (triEGdiME), diethylene glycol diethyl ether (diEGdiEE), ethylenethiourea (ETU), sodium selenite (SS), dimethyl phthalate (DMP), naphthalene (NAP), or p-nitrophenol (PNP) were administered by gavage for eight consecutive days to female CD-1 mice. Weight loss was insensitive as an index of sublethal adult toxicity and was inadequate for determining a maximum tolerated dose. LD50 values indicate that SS, NAP, and PNP were more toxic (8.4, 353.6, and 625.7 mg/kg, respectively) than the polyglycol ethers, ETU, and DMP (LD50 values ranged from 2525.8 to 6281.9 mg/kg). Each of the compounds was administered on d 7 through 14 to pregnant animals at a single dose estimated to be at or just below the threshold of adult lethality. In such a reproductive study, each of the compounds could be categorized on the basis of the pattern of maternal lethality and fetotoxicity which it produced. The number of dams with complete resorptions was significantly increased after administration of ETU, and no mice in the EGdiME-, diEGdiME-, or triEGdiME-treated groups delivered any viable offspring. Maternal lethality was significant in the EGdiME, triEGdiME, PNP, and NAP groups. There was a slight reduction in the average number of live pups per litter in the diEGdiEE- and PNP-treated groups and a significant reduction in the NAP group. The number dead per litter was increased with diEGdiEE. SS and DMP had no effect on maternal or fetal survival at the doses administered. Individual pup weight at d 1 postpartum was only significantly reduced by diEGdiEE, and no gross congenital abnormalities were detected in neonates from any treatment group. These results provide guidelines for the subsequent toxicity testing of these chemicals.

The relationship between prenatal lethality or fetal weight and intrauterine position in rats exposed to diethylstilbestrol, zeranol, 3,4,3',4'-tetrachlorobiphenyl, or cadmium.

When administered orally during gestation, diethylstilbestrol (DES), zeranol (ZN), and 3,4,3',4'-tetrachlorobiphenyl (4CB) but not cadmium (Cd) exhibited significant developmental toxicity, including elevated embryo- and fetolethality and reduced fetal weight, in Sprague-Dawley rats. An analysis was performed to determine the effect of intrauterine position on these parameters. In control dams sacrificed after day 18, the general pattern was that fetuses at the ovarian end of the uterine horns were significantly lighter in weight, while the heavier fetuses were located in middle positions. Treatment with each of the chemicals reduced fetal weight equally across all uterine positions. An inverse of the weight pattern was observed for prenatal mortality in controls. Embryonic resorptions were relatively more frequent at both ovarian and cervical ends, while conceptuses at intermediate positions were less vulnerable. No significant alterations in this pattern were observed in treated litters. The frequency of late fetal deaths in 4CB-treated litters was significantly higher at the cervical end of the horn, however. No differences between horns or between sexes were observed in the relative position patterns for either weight or mortality.

Different patterns of developmental toxicity in the rat following prenatal administration of structurally diverse chemicals.

Differences in the profiles of developmental toxicity for four structurally diverse chemical compounds have been defined following prenatal exposure in the rat. Diethylstilbestrol (DES), 3,4,3',4'-tetrachlorobiphenyl (4CB), zeranol, and cadmium were administered by gavage to Sprague-Dawley rats daily from d 6 through d 18 of gestation. Dams were sacrificed at four prenatal endpoints and the numbers of live and dead fetuses and resorbed embryos were counted. Additional dams were allowed to bring their litters to term, and their offspring were monitored until they reached adulthood. DES induced prenatal death primarily in early embryonic life, and also during parturition. 4CB increased mortality from late in gestation up to 24 h after birth, and altered the sex ratio of survivors by selectively acting against males in utero. Exposure to zeranol resulted in embryolethality only. Cadmium was not lethal to the conceptus at any dose below the dose that caused maternal mortality. Only 4CB had an obvious teratogenic effect, causing intestinal hemorrhage. All compounds produced transient perinatal decreases in the weight of the offspring.

The effects of prenatal exposure to structurally diverse chemicals on the ontogeny of rat dehydrogenases.

Activity levels of sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH) were measured in the livers and brains of rats treated prenatally with 3,4,3',4'-tetrachlorobiphenyl (4CB, 3 mg/kg/day), diethylstilbestrol (DES, 10 micrograms/kg/day), zeranol (ZN, 4mg/kg/day), and cadmium (Cd, 25 mg/kg/day) and compared with enzyme levels for control groups. Enzyme activities were measured at days 15, 17, 19, and 21 prenatally, and days 1, 5, 10, 21, 35, and 56 postnatally. SDH activity was not altered by treatment with 4CB, DES, or ZN, but Cd produced reduced levels in both liver and brain of sexually mature offspring. The patterns of LDH and G6PDH, including sexual differentiation of the latter in adult liver, were not affected by any of the treatments in either tissue. The developmental profiles of each of these enzymes in untreated animals is unique, suggesting that a similar catalytic mechanism is not a factor in determining the patterns of their developmental accumulation.

Delay in the onset of parturition in the rat following prenatal administration of developmental toxicants.

The duration of gestation was determined in Sprague-Dawley albino rats exposed during days 6-18 of pregnancy to diethylstilbestrol (DES) or 3,4,3',4'-tetrachlorobiphenyl (4CB). Each compound produced a delay in the onset of parturition compared to corn-oil-treated controls. The incidence of perinatal mortality was significantly elevated in animals born after 22 days of gestation in both control and treated litters.

Induction of prenatal toxicity in the rat by diethylstilbestrol, zeranol, 3,4,3',4',-tetrachlorobiphenyl, cadmium, and lead.

A teratological study was conducted in pregnant Sprague-Dawley rats dosed orally with diethylstilbestrol (DES), zeranol (ZN), 3,3',4,4'-tetrachlorobiphenyl (4CB), cadmium, or lead on days 6-18 of gestation. Fetuses were examined on day 19 for growth retardation, resorption, gross malformations, and organ-level anomalies. Synthesis of protein, DNA, and proteoglycan in fetal limb cartilage was also studied by measuring the incorporation of labeled precursors in vitro. DES, ZN, and 4CB produced a dose-dependent increase in embryolethality. Treatment with DES caused an increase in cryptorchidism and edema, and reduced average fetal weight. Zeranol also decreased fetal weight, but was not teratogenic nor did it alter rates of synthesis of macromolecules in cartilage. 4CB caused severe intestinal lesions that were associated with accumulation of blood in the digestive tract and amniotic fluid. 4CB also decreased overall fetal size and total and ossified tibia lengths. Cadmium produced no malformations although incorporation of [3H]amino acids by limb cartilage was slightly increased. Lead was not teratogenic.

Indicators of developmental toxicity following prenatal administration of hormonally active compounds in the rat. I. Gestational length.

Prenatal administration of 3,4,3',4'-tetrachlorobiphenyl, zeranol, or diethylstilbestrol was observed to delay parturition in the rat and result in a concomitant increase in perinatal mortality. Even among control animals, those litters in which birth occurred after the beginning of day 22 of pregnancy contained significantly fewer survivors one day after birth. Increases in the length of gestation were correlated with increased weight of newborn control pups independent of litter size. The relationship between weight and litter size was anomalous, however, in treated animals. Gestational length is a sensitive indicator of a developmental effect even in the absence of overt teratogenicity.