Modification of the complement induced by PGE2 and gonadotropins

The Complement is one of the major effectors of the humoral aspecific immune system building up a defence mechanism of the organism. As it is known that some hormonal substances like gonadotropin (hCG) and some hormone-like substances (PGE2) influence the entire immunitary system, we wanted to see if they had specific action on the Complement. The measurement of CH50 was carried out using Mayer's method, derived by Ferrazzi and modified by us. Fractions C3 and C4 were measured by means of immunochemistry using Beckman nephelometer. The treatment with hCG (1,000 U + 10 Lf tetanus toxoid) caused an increase in the CH50 and in the fraction C3, while the fraction C4 was not modified. The treatment with PGE2 (0.25 microgram/rat/die) caused a higher increase of CH50 and C3 fraction. It seems possible to acknowledge C3 involvement in the variation of the Complement's haemolytic activity and this could confirm the intervention of the "alternative pathway". The notable increase in the activity of the Complement induced by hCG and PGE2 could indicate an alternative mechanism of activation of the aspecific humoral immunity in the defence of the organism in all those physio-pathological situations where these substances cause a state of depression of cellular mediated activity.     

Pre-clinical evaluation of an sLe x-glycosylated complement inhibitory protein in a non-human primate model of reperfused stroke

BACKGROUND: Soluble complement receptor-1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x-glycosylation of sCR1. In an effort to translate sCR1-sLe x to clinical trials, we evaluated this agent in a primate stroke model. METHODS: Adult male baboons randomly received either sCR1-sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH50) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post-ischemia. RESULTS: The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH50 levels were significantly reduced in the sCR1sLe x-treated groups. A hypotensive response was also observed in animals treated with sCR1-sLe x. Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1-sLe x.     

Estradiol effect on indomethacin and prostaglandin E2-modulation of adrenergic transmission in rabbit oviduct.

The effect of prostaglandin E2 /PGE2/ and indomethacin on 3H-noradrenaline (3H-NA) release- and on contractions-evoked by field electrical stimulation (FES) was studied in vitro in oviductal isthmus of mature rabbits (untreated and treated with estradiol). FES evoked guanethidine-sensitive contractions and calcium-dependent tritium overflow, which reflected 3H-NA overflow. Marked and concentration-dependent decrease of FES-evoked contractions by PGE2 (0.1-100 nM) was observed in both groups of animals. The inhibitory effect of PGE2 was more pronounced in estradiol treated animals (IC50 1.5 nM, n = 9) than in untreated animals (IC50 18 nM, n = 6). Indomethacin, 1 microM, induced a remarkably pronounced increase of FES-evoked contractions in estradiol treated (by 57.3 +/- 6.3%, n = 8) in comparison with untreated rabbits (21.4 +/- 3.8%, n = 7). The amount of FES-evoked release of tritium was significantly higher in untreated than in estradiol treated rabbits. PGE2 decreased and indomethacin increased tritium-evoked release. The effects of PGE2 and indomethacin on tritium-evoked release showed no estradiol dependence. The competitive results of PGE2 and indomethacin on both evoked contraction and 3H-NA release suggest that endogenous prostaglandin E2 takes part in modulation of adrenergic mediated contraction and that estradiol enhanced the prostaglandin effect.     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.     

Accelerated ovum transport in rabbits induced by endotoxin 1. Changes in prostaglandin levels and reversal of endotoxin effect

The effect of endotoxin (Salmonella enteritidis-Boivin) on ovum transport in the rabbit was examined. A dose of 10 μg/kg intravenously (iv) given 24 h after an injection of human chorionic gonadotrophin (hCG) to induce ovulation caused expulsion of 87% of ova from the oviduct within 24 h. The ED₅₀ and 95% probability limits were 3.1 (2.38–4.03) μg/kg. A dose of 20 μg/kg given at 24 h after hCG exerted its effect on ovum transport within 4 h. Concurrent treatment with indomethacin completely prevented the effect of endotoxin on ovum transport. Endotoxin caused an increase of prostaglandin-like material (PG) E, measured by radioimmunoassay, in uterine vein blood within 35 min and PGE levels continued to rise until 3 h after endotoxin and remained elevated until 8–9 ½ h. PGF in uterine vein blood was not elevated until 90 min after endotoxin and then increased more rapidly than PGE during the next 2.5 h: it was still elevated at 8–9 ½ h. The ratio of PGF:PGE in uterine vein blood decreased from 3:1 in 24 h control samples to 1:1 at 1 h after endotoxin, and then increased rapidly exceeding 5:1 at 2 h. In animals given both indomethacin and endotoxin PG levels in uterine vein blood declined. Phenoxybenzamine partially prevented the effect of endotoxin on ovum transport and in animals so treated PGE levels in uterine vein blood increased similarly to those in animals receiving endotoxin alone, but PGF values, while elevated, were suppressed compared to those in endotoxin animals and the PGF:PGE ratio never exceeded 2:1. It is concluded that endotoxin induces accelerated ovum transport by causing an initial relaxation of the oviductal isthmic musculature due to PGE dominance followed by stimulation of oviductal circular musculature due to PGF dominance.     

Effects of protein synthesis inhibition on PGE2 production in rabbit colonic explants cultured in vitro

Colonic mucosal biopsies cultured for 6 h in the presence of cycloheximide (CH) showed a dose-dependent inhibition of protein synthesis but a biphasic PGE2 production pattern with an increase in both basal and A23187 stimulated PGE2 release at 0.2 microM. At 10 microM CH both protein synthesis as well as basal and PMA induced PGE2 production was inhibited by 90% whereas A23187 stimulated release showed a 50% decrease. At a dose of 100 microM, CH totally blocked also A23187 stimulated PGE2 release without much further decrease in protein synthesis. The effects of 10 microM CH were time-dependently reversible. In biopsies loaded with 3H-arachidonic acid (AA), 10 microM CH had no apparent effect on phospholipase A2 activity, nor could exogenous AA overcome the CH inhibition of basal PGE2 release. No inhibition of prostaglandin synthetase (PS) activity was found in homogenates of biopsies treated with 10 microM CH for 6 h. No direct effect of CH (up to 1 mM) was seen in control homogenates. It is concluded that at least one step in the PGE2 production is protein synthesis dependent. The effect is however not due to a limitation in the enzymes of the major PS system but more likely to one of its co-factors. This factor only plays a role in the intact cell and its importance seems to be reduced during A23187 conditions possibly due to altered cell status and/or other sources of PS. Commonly used high doses (100 microM) of CH give unspecific effects unrelated to inhibition of protein synthesis.     

Relationship between in vivo and in vitro 17 alpha-hydroxylase and C17,20-lyase activity in ovaries of immature hypophysectomized rats treated chronically with human chorionic gonadotropin

The production of 3H2O from 17 alpha-3H-progesterone and 14CH3COOH from [21-14C]progesterone were used to measure the 17 alpha-hydroxylase and C17,20-lyase activities respectively in the microsomal + mitochondrial fraction of homogenates of ovaries from immature hypophysectomized rats chronically treated with human chorionic gonadotropin (hCG). The highly stimulated thecal and interstitial tissues were considered the only source of enzyme. hCG produced an increase in 17-hydroxyprogesterone, and androstenedione, but a drastic decrease in enzyme activity within 6 h; this could be largely prevented by pretreatment of the rats with cycloheximide or aminoglutethimide but actinomycin D was ineffective. After a nadir at 24 h, enzyme activities increased to more than double those of the starting level; this could be prevented by cycloheximide. Maximal activity levels were greatly decreased by cycloheximide and modestly increased by aminoglutethimide. Cessation of treatment at 60 h followed by a single injection of hCG 24 h later did not cause a loss, but delays of 36 or more hours produced a dramatic decrease in enzyme activity, which could be prevented by aminoglutethimide. The results indicate that the level of activity of these enzymes attained in the ovary following exposure to hCG is determined by a balance between the amount of substrate provided and production of enzyme and/or stimulating factors. Therefore, maintenance of increased enzyme activity induced by gonadotropin appears to be under genomic control.     

Stress-induced testicular hyposensitivity to gonadotropin in rats. Role of the pituitary gland

The time course of stress-induced testicular hyposensitivity to gonadotropins was studied in hypophysectomized or naloxone-treated rats exposed to various periods of immobilization. Blood was collected from a chronically indwelling intra-atrial catheter every hour for luteinizing hormone (LH) and testosterone (T) measurement. Eight hours of immobilization completely suppressed T secretion without significant effect on LH. Human chorionic gonadotropin (hCG, 5 IU/rat, i.m.) induced a marked increase in plasma T levels in normal control groups 3 h post-injection while in immobilized rats the response was completely abolished, even after only 30 min of stress. In hypophysectomized rats, as expected, plasma T levels were undetectable, but, contrary to results obtained in normal animals, hCG induced a similar increase of plasma T levels both in control and stressed rats. Immobilization stress failed to inhibit plasma T values in hypophysectomized rats pretreated for 4 days with human menopausal gonadotropin (hMG) + hCG, while it did so in similarly treated normal animals. Naloxone induced a rise of plasma LH and T levels in control rats, but did not antagonize the stress-induced fall of plasma T concentration. In all groups, steroid testicular content mimicked variations of plasma T values. In particular, in stressed animals the lack of accumulation of testicular 17-hydroxyprogesterone probably reflected a normal activity of 17-20 lyase. These results indicate that stress induces very rapidly a state of Leydig cell hyposensitivity to gonadotropins and a blockade of T biosynthesis. The causal relationship between the two effects is presently not clear but these events seem to be due to stress-induced release of an inhibitory factor of pituitary origin other that endorphin.     

Induction of ovarian follicular cysts in the pregnant rat by human chorionic gonadotropin.

Prolonged stimulation by human chorionic gonadotropin (hCG) induces ovarian follicular cysts in progesterone-synchronized immature rats [Bogovich, Endocrinology 1989; 124:1646-1653]. To determine if unabated stimulation by hCG has a similar effect on follicular development in adult ovaries, pregnant rats were given either 0 (control), 1, or 3 IU hCG twice daily for 9 days beginning on Day 13 of pregnancy. By Day 22 of pregnancy, rats treated with 1 IU hCG possessed large antral follicles at least 1 mm in diameter: approximately 33% larger than the diameters of preovulatory follicles observed in control rats (0 IU hCG). In contrast, rats treated with 3 IU hCG displayed ovarian follicular cysts up to 5 mm in diameter, with well-developed thecae and just a remnant of granulosa cells. Progesterone, androstenedione, and estradiol accumulation was greater in follicular incubates from hCG-treated rats than in incubates from control rats. Progesterone increased in response to cAMP in incubates from all treatment groups on all days tested. Androstenedione increased in response to cAMP on Day 22 of pregnancy for follicles from control animals, on all days tested for follicles from rats treated with 1 IU hCG, and on Days 15-19 for follicles from rats treated with 3 IU hCG. Androstenedione production in the presence of 300 ng of exogenous testosterone was significantly greater in follicular incubates from animals treated with 1 and 3 IU hCG than incubates from control animals on Days 19-22 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post ovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles

In previous studies we have demonstrated that prior to hCG induced ovulation the levels of PGF and PGE in rabbit Graafian follicles increase markedly as ovulation approaches. We have now extended the study to include follicles obtained from animals at ovulation time and up to 48 hours after hCG injection. We have found that PGF reaches a maximum in ovulated follicles at the time of ovulation and then quickly decreases, whereas PGE continues to rise for several hours and then declines. The increase in both prostaglandins is limited to the follicles that actually ovulate. These data further document the proposed role for prostaglandins in the ovulatory process.     

Acute effect of human chorionic gonadotropin (hCG) on 17 alpha-hydroxylase and C17,20-lyase activity in neonatal or prepubertal rat testis

In contrast to the situation in adults, desensitization of androgen production, secondary to loss of enzyme activity, was not found in testes of neonatal rats exposed to human Chorionic Gonadotropin (hCG). In the present study attention was given to the acute effects of a single injection of hCG upon the activity of testicular 17 alpha-hydroxylase, C17,20-lyase and the concentration of testosterone in the serum of 5, 10 or 28-30 day old rats was investigated. Tritiated H2O from 17 alpha-[3H]progesterone and 14CH3COOH from 21-[14C]progesterone were the products measured to evaluate hydroxylase and lyase activities respectively. Large increases in hCG in the serum were detected within 2 h of a subcutaneous injection. Testosterone, which was highest in 5 day animals, increased quickly in all animals given hCG. In 28-30-day old animals, the concentration of this steroid began to fall 24 h after injection of hCG. 17 alpha-Hydroxylase activity decreased in the testes of all animals given hCG, but only after a brief increase. Activity returned to the starting level, or above, within 24 h in 5 or 10-day old animals. In 28-30-day old rats the activity of both enzymes decreased dramatically to a nadir at 24 h, but increased thereafter. The results indicate that desensitization of testicular androgen synthesizing enzymes occurs in neonatal as well as older testes stimulated with hCG, but the desensitization was very brief in neonatal animals and no desensitization of lyase was found in 5-day old rat testes.     

Sensitivity of mice to systemic hyperthermia: effect of ascites tumor cell transplants

The effect of a transplantation of mastocytoma cells in the abdominal cavity on the sensitivity of mice to a systemic hyperthermia was studied. The systemic hyperthermia was induced by exposing whole-body of animals to 2,450 MHz waves under anesthesia. Core body temperature was raised up to 42.0 +/- 0.2 degrees C in 15 min and maintained constant at the temperature for variable length of time. Thermosensitivity of animal was expressed with LD50, 42 degrees which was the length of heating time at the temperature of 42 degrees C lethal for 50% of the animals examined. The transplants were mastocytoma FMA3 cells. They were transplanted at a dose of 10(5) cells per mouse. The LD50, 42 degrees observed 3, 12 hrs, 1, 2, 3 and 6 days after the transplantation was 33, 23, 17, 24 and 35 min, respectively. In mice without tumor it was 43 min.     

Adipose differentiation-related protein: a gonadotropin- and prostaglandin-regulated protein in primate periovulatory follicles

The midcycle LH surge stimulates a rise in follicular fluid prostaglandin E2 (PGE2), which is necessary for normal ovulation. To examine PGE2-regulated processes in primate follicles, monkey granulosa cells were cultured with hCG alone or with hCG and PGE2, and the resulting total RNA was subjected to microarray analysis. Twenty PGE2-regulated mRNAs were identified, and we selected a lipid droplet protein, adipose differentiation-related protein (ADRP), for further study. To determine whether hCG and PGE2 regulate ADRP expression in vivo, monkeys received gonadotropins to stimulate multiple follicular development. Human chorionic gonadotropin was then administered alone or with the PG synthesis inhibitor celecoxib, and follicular aspirates or whole ovaries were obtained at times that span the 40-h periovulatory interval. Administration of hCG increased granulosa cell ADRP mRNA and protein, with peak levels measured just before the expected time of ovulation. Treatment with hCG and celecoxib decreased granulosa cell ADRP mRNA levels compared with those of animals treated with hCG only. ADRP was detected by immunocytochemistry in many monkey tissues that synthesize prostaglandins but was not consistently expressed by steroidogenic tissues. Granulosa cells of periovulatory follicles immunostained for ADRP after, but not before, hCG administration; ADRP colocalized with large lipid droplets within the granulosa cell cytoplasm. These studies identify ADRP as a novel gonadotropin- and PGE2-regulated protein in the granulosa cells of primate periovulatory follicles. Because ADRP facilitates arachidonic acid uptake in non-ovarian cells, ADRP-associated lipid droplets may enhance arachidonic acid uptake by granulosa cells to provide a precursor for periovulatory prostaglandin production.     

Serum FSH, LH and testosterone in the male rhesus following prostaglandin injection.

Adult male rhesus were treated with PGE2, PGF2 alpha or the 13,14-dihydro-15-keto metabolite of PGE2 in a randomized crossover design. Serum concentrations of FSH, LH and testosterone were determined and compared to the respective values in the same uninjected animals. No significant changes were noted in controls or following the metabolite injection. FSH increased gradually for 4 hours after metabolite treatment. In contrast, injection of PGF2 alpha was followed by an abrupt (within 15 minutes) increase in LH and testosterone. FSH increased gradually in 2 of 3 treated animals. Injection of PGE2 was followed by a similar abrupt increase in LH concentration. This was not always associated with a significant increase in testosterone or FSH. These results demonstrate that injections of PGE2 or PGF2 alpha can change serum gonadotropin and testosterone concentrations in male rhesus monkeys, and that the effects of these two prostaglandins are qualitatively different.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transplantable rat Leydig cell tumor--5. Cellular localization of beta-adrenergic and prostaglandin receptors and their effects on testosterone production

The cellular localization of beta-adrenergic and prostaglandin (PG) receptors and their effects on adenylate cyclase activity (AC) and testosterone production in vitro were investigated in a transplantable rat Leydig cell tumor (H-540). Separation of the tumor cells in Percoll gradients revealed that the specific binding of [3H]PGE1 and [125I]Cyanopindolol was found in the same fraction as that of [125I]LH. This fraction--judged by light microscopy of smears--consisted of tumor Leydig cells. In addition, [125I]cyanopindolol was found specifically bound in the red blood cell fraction. In the Leydig tumor cells, approx 25% of the beta-adrenergic receptors was identified as beta 1-receptors, whereas approx 75% of the receptors were of the beta 2-subtype. The AC in Percoll purified Leydig tumor cells was stimulated by hCG (6-fold), PGE1 (2-fold), PGE2 (1.5-fold), PGI1 (2-fold) and isoproterenol (2-fold). The AC in the red blood cell fraction was stimulated by isoproterenol whereas the PGs and hCG had little or no effect. hCG, isoproterenol and PGE1 were able to stimulate testosterone production in vitro. At 44 h incubation, PGE1 was the most potent stimulator of testosterone production. In conclusion, tumor Leydig cells possess hCG, PGE1, PGI2 and beta-adrenergic receptors coupled to the AC. PGE1 and beta-adrenergic agonists stimulate testosterone production after prolonged incubation in vitro.     

In vitro prostaglandin production by bovine corpora lutea destined to be normal or short-lived

Basal and calcium ionophore (CaI)-influenced production of prostaglandins (PGs) by corpora lutea (CL) destined to be normal or short-lived were compared. Ovulation was induced in 24 lactating beef cows with human chorionic gonadotropin (hCG, 1000 IU) administered between 35 and 40 days postpartum. Ten cows received norgestomet implants for 9 days prior to induced ovulation (Normal CL) and 14 served as untreated controls (Subnormal CL). Five cows in each treatment were unilaterally ovariectomized on Day 6 (Day 0 = day of hCG administration) and CL were collected. Blood samples were collected daily through-out the experimental period from cows not ovariectomized. Plasma progesterone (P4) in ovary-intact animals indicated that short-lived CL were induced in 8/8 cows not pretreated with norgestomet, and normal luteal lifespan was observed in 4/5 implanted cows. Dispersed luteal cells were incubated for 8 h with 0, 0.05, 0.5, or 5 microM CaI (A23187). Incubation media were analyzed for P4, PGF2 alpha, 6-keto-PGF1 alpha (PGI), and PGE2. The weight, cell number, and basal or CaI-influenced production of P4 did not differ between Normal CL and Subnormal CL. Basal production of PGF2 alpha, PGI, and PGE2 was higher in Subnormal CL than in Normal CL (p less than 0.05). In response to 0.05 microM CaI, PGF2 alpha was stimulated in Subnormal CL (p less than 0.01), while PGI (p less than 0.05) and PGE2 (p less than 0.1) were increased in Normal CL. Production of PGs was reduced by 5 microM CaI in Subnormal CL (p less than 0.01), but not in Normal CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of polyunsaturated fatty acids and gonadotropin on prostaglandin series E production in a primary testis cell culture system for the European sea bass

Prostaglandin series E (PGE) production by mixed populations of isolated sea bass Dicentrarchus labrax testicular cells was measured after culture at 20° C in L-15 for up to 24 h in the absence or presence of arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and/or gonadotropin (hCG). AA stimulated a significant dose- and time-dependent increase in PGE production. EPA stimulated PGE production only during the first 6 h although levels were 26·4% those induced with AA; DHA reduced PGE production. Although hCG alone had no effect, it enhanced AA-induced PGE formation and suppressed EPA-induced PGE production. These results suggest that essential PUFAs modulate significantly testicular PGE production in vitro and therefore may have important effects on steroidogenesis and spermiation in the male European sea bass.     

Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3, cs70. II. Brain.

Most cell divisions in the mouse brain have ceased by 14 days after birth. Therefore, spontaneous mutations that occur in brain cells can be assumed to be fixed by replication during brain development. Spontaneous and ethylnitrosourea (ENU)-induced reverse mutations at a single AT base pair were measured in brain tissue by using mice transgenic for PhiX174 am3, cs70. The line (am54) has 50 PhiX genomes per haploid genome integrated in a tandem array and is maintained by random breeding on a C57BL/6 background. For mutagenesis studies, homozygous am54 males were mated to non-transgenic C57BL6/J females. Four-day old offspring from this cross were treated with 50 mg/kg ENU and were euthanized at 68-80 days of age. The ENU-treated animals had a significantly higher frequency of am3 revertants in brain than did concurrent controls. In a second experiment, hemizygous male offspring (85 to 94 days old) were treated with 150 mg/kg ENU and euthanized at various post-injection intervals (3, 10 and 110 days). The revertant frequencies 3 and 10 days after treatment were not significantly different from control values. At the 110 days post-injection interval, however, the average revertant frequency in the treated group was significantly lower than controls. In a second study animals were euthanized 3, 10 and 74 days after treatment. Two groups (3 and 74 days post-injection) also showed a significant decrease in the revertant frequency as compared to controls. Additional sets of adult animals were treated with 50 and 150 mg/kg ENU and were euthanized 195 to 201 days after treatment. The average revertant frequency of the animals that were treated with 50 or 150 mg/kg ENU was not significantly different from the control value. Thus, although an increase in mutant frequency is detected in the PhiX174 system when neonatal mice are treated with ENU, treatment of mature mice with ENU did not result in an increase in the mutant frequency. Moreover, under certain conditions, ENU-produced a significantly lower mutant frequency than was observed in the control animals. This decrease in the revertant frequency among the treated animals was likely due to selective killing of cells with a higher spontaneous revertant frequency than cells with lower frequency.