An overview of the influence of ACE inhibitors on fetal-placental circulation and perinatal development.

The renin-angiotensin system is associated with a variety of pathophysiological processes in many organ systems, and is known to be involved in the normal regulation of blood pressure and in the pathogenesis of renovascular hypertension. Angiotensin II is a multifunctional hormone that manifests its properties by interacting with two major subtypes of cell surface receptors (AT1 and AT2). Angiotensin converting enzyme (ACE) inhibitors are able to modify the actions of the renin-angiotensin system, and are indicated for the treatment of hypertension and heart disease. The antihypertensive effects of ACE inhibiting drugs are related to their ability to block the conversion of the decapeptide, angiotensin I, to the potent pressor octapeptide, angiotensin II. ACE inhibitors have been implicated in fetopathies in humans and perinatal mortality in rats, rabbits, sheep and baboons. Human fetopathies were seen when ACE inhibitors were given around the 26th week of gestation. The major adverse effects in babies include: oligohydramnios, renal tubular dysgenesis, neonatal anuria, calvarial and pulmonary hypoplasia, mild to severe intrauterine growth retardation, persistent patent ductus arteriosus and fetal or neonatal death. These developmental anomalies are thought to be partly due to a direct action of ACE inhibitors on the fetal renin-angiotensin system and partly due to the ischemia resulting from maternal hypotension and decreases in fetal-placental blood flow and oxygen/nutrient delivery to the fetus. The purpose of this review is to briefly discuss the pathophysiological role of the renin-angiotensin system, the therapeutic uses of ACE inhibitors in pregnant patients and to focus primarily on the major fetotoxic effects of ACE inhibitors encountered in humans and animal models. I will also review our recent data which show that capozide (captopril + hydrochlorothiazide) not only produces oligohydramnios but also disturbs the balance of glucose and NaCl in the maternal plasma and amniotic fluid of the rat.

Developmental toxicity of 4-substituted amphetamines in mice.

Despite overwhelming and tragic evidence of their detrimental and dangerous consequences, amphetamines remain significant drugs of abuse and addiction. The effects of 4-substituted amphetamines: 4-hydroxyamphetamine (4-HA), 4-methoxyamphetamine (4-MEA), 4-ethoxyamphetamine (4-ETA), 4-propoxyamphetamine (4-PPA), and 4-benzyloxyamphetamine (4-BEA) on intrauterine development, pregnancy outcome, postnatal growth, and survival were compared in Swiss-Webster mice. Single daily doses (0, 50, or 100 mg/kg) of an aqueous solution of different amphetamines were administered on pregnancy days 6 through 18. The 50 mg/kg doses of all amphetamines were well tolerated by the mothers and did not produce any overt signs of maternal toxicity or death. However, a few mothers died on different days of gestation after receiving 100 mg/kg of 4-HA, 4-MEA, 4-ETA, and 4-BEA. The mothers that failed to deliver naturally (3 d after the due date) were killed and their uteri were examined for live/dead fetuses and resorption sites. In comparison with respective controls, the incidence of resorptions was markedly higher in the 4-MEA- and 4-ETA-dosed groups. Delivery was prolonged in the 4-PPA- and 4-BEA-treated dams. Apparently well-formed but dead pups were delivered by 4-HA-, 4-PPA-, and 4-BEA-dosed mice. Marked reductions in average litter size and weight occurred after intrauterine exposure to 100 mg/kg 4-BEA. Treatment with 4-ETA, 4-PPA, and 4-BEA not only resulted in a high incidence of cannibalism within 24 h after birth but also caused an increase in cumulative pup mortality during the first 3 weeks of age. Body weight gain was significantly lower in 3-week-old offspring exposed to 4-HA and 4-PPA than in the controls. The findings suggest that 4-substituted amphetamines exhibit a wide variation in their effects on maternal toxicity and pregnancy wastage, and produce adverse effects on parturition, pup survival, and postnatal development.

The influence of pregnancy on the biotransformation and urinary excretion of methoxyphenamine in mice.

(i) The urinary elimination of methoxyphenamine (MPA) and its metabolites in underivatized samples was examined after single and multiple oral administration to pregnant and non-pregnant mice by GLC and GLC-MS. (ii) The major metabolite O-desmethylmethoxyphenamine (ODMP), along with lesser amounts of N-desmethylmethoxyphenamine (NDMP) and 2-hydroxyamphetamine (2OH), were the only metabolites detected in urine extracts of pregnant and non-pregnant mice. 5-Hydroxymethoxyphenamine (5HMP) was not detected. Enzyme hydrolysis did not increase the recovery of either substrate or metabolites in either the pregnant or non-pregnant animals. The results show that MPA metabolism in the Swiss-Webster mouse is distinctly different from that seen in man and other laboratory animals. (iii) The mean MPA:ODMP ratio in day-6 urine from pregnant mice after a single dose was 0.31 +/- 0.04. The NDMP:ODMP ratios were less than 0.10 in all samples. Non-pregnant mice urine had equivalent amounts of MPA, NDMP, ODMP, and 2OH after multiple dosing. (iv) While multiple dosing and pregnancy did not alter either the urinary recovery or profile of the metabolites detected, there was a linear decrease in the MPA:ODMP ratio during gestation. (v) MPA was extensively metabolized to ODMP in the male mice, and the MPA:ODMP ratio of 0.41 was slightly higher than that observed in the pregnant and non-pregnant females.

Evaluation of the rat embryo culture system as a predictive test for human teratogens.

Ingestion of the anticonvulsant drug valproic acid and of the angiotensin converting enzyme inhibitor captopril during pregnancy has been associated with abnormal fetal outcome in humans. In contrast, the use of the antiinflammatory drug ibuprofen and the antihistamine diphenhydramine has not been documented to be embryotoxic in humans. We evaluated the rat embryo culture system as a predictive model of teratogenesis, using these four drugs as test agents. Valproic acid, ibuprofen, and diphenhydramine were embryotoxic, inducing concentration-dependent decreases in growth and a significant increase in anomalies. Valproic acid caused an increase in neural tube defects, ibuprofen increased the incidence of abnormal maxillary processes, and diphenhydramine increased the number of embryos with distorted body morphology. These abnormalities were induced at concentrations of valproic acid and diphenhydramine that are used clinically, but ibuprofen only induced toxicity at concentrations greatly exceeding the therapeutic range. Captopril was not embryotoxic up to 5 mM, the highest concentration tested. These results suggest that the rat embryo culture system produces both false positive and false negative data on the teratogenic potential of drugs. Although such an in vitro assay may be suitable to determine the mechanism of teratogenesis, it is not a sensitive indicator of potential human teratogens on its own. These data support the view that in vitro systems can only supplement clinical and epidemiological observations in humans, possibly as a method to determine mechanisms of actions of teratogens.

Biotransformation and urinary excretion of 4-substituted amphetamines in pregnant mice.

The urinary elimination of 4-hydroxyamphetamine (PHA) and a series of homologous 4-alkoxy-substituted amphetamines and their metabolites was examined after single and multiple oral administration to pregnant and non-pregnant mice. The metabolic profile and extent of biotransformation in a series of alkoxy analogues were affected by the size of the alkoxy side chain, multiple dosing and pregnancy. There were increased recoveries of both the substrate and the conjugated derivative of PHA during gestation. The major metabolic routes observed were O-dealkylation, conjugation, and aliphatic hydroxylation of the propoxy side chain. There was some evidence of oxidative deamination. Pregnancy did not alter the profile of the major metabolites detected by GLC and NMR spectroscopy.

Effect of ciprofloxacin on the disposition of 14C-dideoxyinosine-derived radioactivity in the rat.

The potential for the antibacterial agent ciprofloxacin to interfere with the disposition of the antiviral agent dideoxyinosine (ddI) was evaluated in adult male Wistar rats. The combination group (n = 10) was dosed orally with 100 mg/kg ciprofloxacin in water every 12 h for three consecutive doses. Thirty minutes after the last dose, a bolus IV injection of 50 mg/kg 14C-ddI in physiologic saline was given over 7-10 sec through the penile vein. The control group (n = 8) received a single dose of IV 14C-ddI. Blood, urine and fecal samples were collected from 0-4 h, and tissue samples were removed at 4 h after ddI administration. The disappearance of 14C from blood followed the kinetics of a two-compartment model with bolus input and first-order output. The following pharmacokinetic parameters (mean +/- SD) were calculated based on total blood radioactivity for the 14C-ddI alone and combination groups, respectively: blood clearance 13.5 +/- 2.0 vs. 12.7 +/- 1.5 ml/min/kg, terminal disposition half-life 49.9 +/- 2.2 vs. 49.0 +/- 6.0 min, steady-state volume of distribution 891 +/- 134 vs. 825 +/- 126 ml/kg, mean residence time 66.2 +/- 3.1 vs. 65.0 +/- 7.8 min (the power to detect a 20% decrease in the above parameters, relative to the reference ddI alone, was > 85% at the 5% significance level), percentage of administered dose recovered in urine 62.2 +/- 9.8% vs. 56.6 +/- 13.0%. Most of the radioactivity in tissues was concentrated in the liver, adrenal, spleen and kidney. Except for significant decreases (p < 0.05) in the levels of 14C in skeletal muscle and cecum of combination treated rats, the coadministration of ciprofloxacin showed no differences in either the tissue levels of 14C or the pharmacokinetic parameters of 14C-ddI-derived radioactivity. The results obtained with total 14C measurements suggest that ciprofloxacin does not alter the disposition of ddI in the rat, although unchanged ddI was not determined.

In vitro effects of 'designer' amphetamines on human peripheral blood mononuclear leukocytes proliferation and on natural killer cell activity.

Human peripheral blood mononuclear leukocytes (PBML) proliferation was measured in the presence or absence of amphetamines. Proliferation in response to T-cell mitogen PHA was suppressed from 22 to 34% by d- and dl-amphetamine, respectively, contrarily to 1-form which did not affect proliferation of PHA-stimulated PBML. The 'designer' amphetamines appeared to be more potent inhibitors of PBML proliferation induced by both PHA and PWM stimulation than those of the racemic and isomeric forms of amphetamine. A wide variation was seen in the suppressive actions of the 'designer' amphetamines, and the mean percentages of suppression varied from 12 to 45% compared with the control values. 4-Propoxy-amphetamine (4-PA) was found to be the most active among the 'designer' drugs. In vitro effects of d-, 1- and dl-amphetamine were also studied on natural killer (NK) cell activity. A marked increase in the NK cell activity was observed only in the presence of very low concentrations (10(-12) to 10(-10) M) of dl-amphetamine, however, the activity of the NK cell remained within the control limits in the presence of d- or 1-forms. The findings suggest that the abuse of amphetamines, especially the 'designer' drugs, may adversely affect the activity of immunoregulatory cells and might lead to a compromised immune system in amphetamine abusers.

Lithium-induced nephrotoxicity in rats following subcutaneous multiple injections and infusion using mini-osmotic pumps.

The nephrotoxicity of the anti-manic-depressive drug lithium (Li) is well recognized but the effects of fluctuation in plasma levels from different Li dosage regimens are not. Experiments were done to compare the nephrotoxicity of Li in rats treated either with subcutaneous multiple injections (SMI) or by infusion using mini-osmotic pumps (MOP) vs concurrent controls. A dose of 2 meq/kg/day of LiCl dissolved in saline was given for 8 consecutive days. During treatment, the animals were kept in individual metabolism cages and their plasma Li levels, water intake, urine volume, pH, and osmolality were monitored daily. In the SMI group, average daily plasma Li levels, 30 min post-injection and just prior to the next injection, were 2.6 and 0.34 meq/liter, respectively. The daily mean plasma Li level in the MOP group was 0.39 meq/liter. While both Li treatment groups showed evidence of toxicity not seen in the respective controls, overall differences in the nephrotoxic response between the Li regimens emerged on Days 6 to 8. Thus water consumption and urine volume were both raised with SMI and by Day 8 the urine osmolality was reduced (all p less than 0.05). The results indicate that after administering equivalent amounts of Li, SMI treatment produced a higher degree of nephrotoxicity than the MOP treatment.

A comparative study of the pharmacokinetics of propranolol and its major metabolites in the rat after oral and vaginal administration.

1. The concentrations of propranolol (PPL) and its metabolites were monitored by h.p.l.c. in serum of rats during the first 6 h after administering single doses (20 mg/kg) of PPL either orally or intravaginally (i.vg). 2. The results showed that PPL was quickly transferred to the systemic circulation from the rat vagina and the serum concentration profile as substantially altered by the route of administration. Serum concentrations of free PPL were significantly higher in i.vg-dosed animals than their oral dosed counterparts. 3. Inter-animal serum conc. variations of PPL and its metabolites in the i.vg-dosed rats were smaller than those of the orally treated females. 4. In comparison with the i.vg-dosed rats, the levels of PPL metabolites (propranolol glycol, naphthoxylactic acid, naphthoxyacetic acid) were greater by the oral route, though these differences were not statistically significant. 5. The serum elimination half-lives (t1/2)beta of PPL and its metabolites during the beta-phase were not significantly different in the two treatment groups. 6. Following i.vg application, both the AUC and the Cmax values of PPL were significantly greater than those of orally dosed females, while no statistically significant differences were found in the tmax values. 7. Comparison of the AUC values showed that relative bioavailability of PPL was approx. 36 times greater in i.vg-treated animals than those of the orally dosed rats.

Effects of in utero exposure to cyclophosphamide in mice. I. Pregnancy outcome and observations of offspring during early postnatal life.

Swiss-Webster mice treated orally with cyclophosphamide (Cy) (1, 2.5 or 5 mg/kg) once daily on gestational days 6 through 18 gave birth to pups which appeared to be normal and the majority of which survived to adulthood. The maternal treatment resulted in reduced mean pup weight at birth and increased cumulative pup mortality. On the other hand, pregnancy outcome and mean pup body, spleen and thymus weights, when measured at 4 weeks of age, were within the control ranges. Similarly, hematological profiles, serum immunoglobulin (IgG, IgM) levels and histology of the lymphoid tissue (spleen and thymus), assessed at 4 weeks of age, were not affected by the maternal treatment. Treatment with 7.5 mg/kg Cy not only resulted in reduced litter size but also increased the cumulative pup mortality. In addition, with this dose, histopathological changes were observed in the thymus in 2-and 3-week old pups. The morphology of the thymus in 4-week old pups was unremarkable. At the dose of 10 mg/kg no live births were recorded.

Propranolol metabolism by Cunninghamella bainieri.

1. Incubations of racemic propranolol alone or in the presence of either quinidine or sparteine were performed with Cunninghamella bainieri. 2. Five mammalian metabolites of propranolol (4-hydroxypropranolol, desisopropyl-propranolol, 1-naphthoxylactic acid, propranolol glycol and 1-naphthoxyacetic acid) were present in unhydrolysed extracts of the incubation medium according to h.p.l.c. and g.l.c. analyses. The relative proportion of 4-hydroxypropranolol increased after enzymic treatment. 3. Propranolol not only had a fungistatic effect, but also caused morphological changes in the organism, which were accompanied by decomposition of 4-hydroxypropranolol and formation of a greenish-brown colour in the incubation medium. 4. Drug interaction experiments yielded results which paralleled those reported in mammals. 5. The findings indicate that C. bainieri may be a useful microbial model for drug disposition and interaction studies.

Effects of in utero exposure to cyclophosphamide in mice. II. Assessment of immunocompetence of offspring from 5 to 10 weeks of age.

Offspring of mice treated with cyclophosphamide (Cy; 1, 2.5 or 5 mg/kg) during pregnancy (6-18 days of gestation) and tested for immunocompetence from 5 to 10 weeks of age were found to have defective reticuloendothelial clearance. The main effects were: a) increased elimination half time (T 1/2) of 51Cr-labeled SRBC from circulation, b) decreased liver uptake of 51Cr and c) impaired ability of the spleen, mostly affecting the female pups, to compensate for decreased liver uptake. The highest dose group suffered the most pronounced effects. This group was also found to have increased IgG immunoglobulin levels at 7 weeks of age. IgG antibody production in response to specific antigenic stimulation and delayed hypersensitivity reactions to oxazolone did not appear to be affected by Cy treatment.

Transplacental and mammary passage of radioactivity in rats treated vaginally and orally with [14C]propranolol.

The milk transfer, maternal-fetal distribution, and disposition of the antihypertensive/spermicidal agent propranolol were studied in pregnant and lactating rats. Single doses (10 mg/kg) of an aqueous solution of [14C]propranolol were administered either orally (po) or intravaginally (ivg) on gestational d 15, or on postpartum d 7-10. Upon ivg administration, [14C]propranolol was quickly transferred to systemic circulation and the mean blood [14C] concentrations were significantly greater during the first 0.25-2 h than in po dosed counterparts. About 98% of the ivg applied dose was absorbed after 6 h in gravid rats, and the combined 6-h excretions of radioactivity in the urine (ivg = 24.6%; po = 22.9%) and feces (ivg = 16.8%; po = 14.6%) were equivalent in both groups. At the end of 6 h, the levels of [14C] in the urinary bladder, adrenal, uterus, ovary, spleen, skeletal muscle, brain, heart, lung and fat were significantly higher in ivg treated rats than po dosed animals. Compared with the maternal plasma (ivg = 0.76; po = 0.88 microgram/ml), the mean concentrations of [14C] in the placentas were similar in both groups, while the amounts of [14C] were three to five times lower in the amniotic fluids and the fetuses of both po and ivg treated dams. In lactating rats, over 99% of the administered radioactivity was absorbed from the vagina within 6 h. The blood concentrations of [14C] were significantly elevated at 0.5 and 1 h in the per vaginam treated animals, and afterward the disappearance rate of [14C] followed a similar course in both groups. Following ivg application, the milk radioactivity peaked at 0.5 h and declined rapidly. However, the appearance of [14C] in milk was rather slow after oral dosing: the milk [14C] peaked between 2 and 3 h posttreatment and remained steady thereafter. The milk to blood (M/B) [14C] concentration ratios were markedly greater during 0.5 to 1 h in the ivg group than in their po dosed counterparts. At 6 h, the [14C] levels in the whole blood, plasma, milk, and mammary gland were virtually equivalent in the ivg and po treated females. Comparison of the areas under the milk [14C] concentration-time curves (AUCs) indicated that the milk availability of [14C] was about 31% more in dams dosed vaginally. These data suggest that route of administration alters the disposition and milk excretion of [14C]propranolol-derived radioactivity in pregnant and lactating rats.

Cytogenetic effects of vaginally administered gossypol in murine bone marrow cells.

The cytogenetic effects of gossypol were evaluated by determining the frequency of sister chromatid exchanges (SCEs), percentage of pulverized metaphases, mitotic indices and micronuclei in bone marrow cells of mice treated per vaginam. A dose-dependent increase in the frequency of SCEs was observed when gossypol suspended in corn oil was administered at dosages of 10, 20 or 40 micrograms/g. In comparison with controls, incidences of SCEs were significantly higher in mice given 20 and 40 micrograms/g gossypol, whereas the mitotic indices, percentages of pulverized metaphases and the frequency of interphase micronuclei in treated animals were not different from their control counterparts. The SCE data suggest that gossypol has a DNA-damaging potency in murine bone marrow cells.

Effects of the spermicide nonoxynol-9 on the pregnant uterus and the conceptus of rat.

In a series of experiments the embryotoxic potential of nonoxynol-9 (N-9) was investigated in adult female rats given a single per vaginam application of 5 mg/100 g (0.1 ml/100 g) of this spermicide on day 3 (pre-implantation period) or 7 (postimplantation period) of gestation. Control rats were given physiologic saline (0.1 ml/100 g) intravaginally. The vulvar labia were apposed for 24 h by metallic clips to prevent leakage of the solution. Groups of dams treated on pregnancy days 3 and 7 were killed by CO2 inhalation on gestational days 6, 9, 12 and 15, and 8, 9, 10, 12 and 15, resorption and total resorption of the conceptus, embryonal and placental resorption and total resorption of the conceptus, embryonal and placental necrosis, placentitis, endometritis, multicystic endometrium, and diffuse or segmental dilatation of the uterine horns. Generally, the incidence of these lesions varied with the length of time after N-9 was administered and it was consistently higher in the females treated on pregnancy day 3 than in those treated on day 7. Acute vaginitis waned with time after the application of N-9. It was concluded that under the conditions of this study, N-9 is embryocidal/fetocidal in the rat if administered during the first week of gestation. The impairment of embryonal/fetal development was attributed to the N-9-induced changes in the endometrium, the placenta and/or the embryo.

Evaluation of the cytotoxicity and genotoxicity of the spermicides nonoxynol-9 and octoxynol-9.

The cytotoxic and genotoxic potentials of the spermicidal agents, nonoxynol-9 (N-9) and octoxynol-9 (0-9), were evaluated in in vitro test systems using rat liver cells. N-9 was also tested for the induction of sperm abnormalities in mice. Dose-related cytocidal effects were seen after the addition of N-9 and O-9 to the culture medium for 24 h. The mean concentrations of N-9 and O-9 necessary to decrease the number of viable cells by 50% (LC50) were 24 and 43 micrograms/ml of media, respectively. The spermicides neither induced DNA repair in freshly isolated hepatocytes, nor caused any mutations at HGPRT locus in the T51B rat liver cell line. There was also a lack of malignant transformation response in the low-calcium assay. Further, the germinal cells of mice remained unaffected by N-9.

PIP: In vitro test systems were employed to evaluate the cytotoxicity and genotoxicity of the vaginal spermicide nonoxynol-9 (N-9) and the related nonionic detergent Triton X-100 (0-9). N-9 is typically used in concentrations of 2-12.5%, or vaginal applications of 50-140 mg, or up to 1 g in the vaginal sponge. Data from rats suggest that N-9 is excreted by the liver and kidneys unmetabolized, but no data on fate of absorbed N-9 are available in women. The tests performed here were the rat liver cell cytotoxicity assay on T51B line cells, the rat liver DNA repair assay using autoradiography with freshly isolated cells, a mutation assay using T51B rat liver cells exposed to azaguanine, a transformation assay with these cells grown in low calcium medium, and a sperm abnormality assay using hybrid male mice. On a molar basis, N-9 was 1.8 times more cytotoxic than 0.9. The LC50s were 24 and 43 mcg/ml respectively. Other work has shown that a level of 100 mcg/ml of N-9 per ml human semen is needed to immobilize sperm within a few minutes. Both detergents exhibited thresholds below which they failed to kill cells. Neither agent had any effect on DNA synthesis, mutation or transformation. No effect was registered on sperm when the detergents were injected ip in male mice at doses up to 60 mg/kg/day for 5 days, although a few mice died from this treatment. Whether the agents reached the testes was not determined.

Induction of sister chromatid exchanges and chromosome damage by gossypol in bone marrow cells of mice.

The chromosome-damaging potential of gossypol was evaluated by scoring sister chromatid exchanges (SCEs), determining the percentage of pulverized metaphases and the mitotic index in bone marrow cells of mice. Bone marrow cells were collected approximately 21 hours after the intraperitoneal (0,20,40,80, or 160 micrograms/g) and oral (0,40,80, or 160 micrograms/g) administration of gossypol acetic acid. Irrespective of the dosing schedule (single or multiple doses), the vehicle used (physiological saline, corn oil, or 10% aqueous ethanol), and the route of administration, the mean SCE count per cell was significantly higher (P less than 0.05) in gossypol-treated groups than their control counterparts. At 80 and 160 micrograms/g dose levels, the occurrence of metaphase chromosome pulverization was significantly greater, while mitotic index values were markedly lower than those of the corresponding control values. The results suggest that gossypol is a potentially mutagenic and clastogenic agent in murine bone marrow cells.

Embryotoxicity of benzalkonium chloride in vaginally treated rats.

The effects of the spermicide benzalkonium chloride (BKC) were studied on the conceptus of rat. Single doses (0, 25, 50, 100 or 200 mg kg-1) of aqueous solutions of BKC were administered intravaginally (1 ml kg-1) on gestational day 1. The vulval metallic clips, used to prevent leakage of the solution, were removed 24 h post-treatment. Fetuses were obtained and examined for malformations on day 21 of gestation. slight to copious amounts of vaginal discharge and vaginitis were noticed in rats treated with the two largest doses of BKC. A dose-related increase in resorptions and fetal death, reduction in litter size and weight were observed in BKC-treated dams. The conceptus loss seemed to occur both before and after implantation. BKC did not cause any discernible visceral malformations, although minor sternal defects occurred in fetuses exposed to 100 and 200 mg kg-1 of the spermicide. These results suggest that single vaginal application of BKC is embryo- and fetocidal in the rat at a dose about 143 times higher than that recommended for controlling conception in women.