In vivo and in vitro investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) on rat embryos

Artemisinin derivatives are clinically effective and safe antimalarials, but are not recommended during the first trimester of pregnancy because of the resorptions and abnormalities seen in animal reproduction studies. Understanding how, when and what toxicity occurs is crucial to any assessment of clinical relevance. Previously, DHA has been shown in the rat whole embryo culture (WEC) to primarily affect primitive red blood cells (RBCs) causing subsequent tissue damage and dysmorphogenesis. To verify the primary target of DHA in vivo and to detect consequences induced by early damage on embryo development, pregnant female rats were orally treated on gestation days (GD) 9.5 and 10.5 with 7.5 or 15 mg/kg/day DHA and caesarean sectioned on GD11.5, 12.5, 13.5, 15 and 20. A parallel in vitro WEC study evaluated the role of oxidative damage and examined blood islands and primitive RBCs.

In accordance with the WEC results, primitive RBCs from yolk sac hematopoiesis were the target of DHA in vivo. The resulting anemia led to cell damage, which depending on its degree, was either diffuse or focal. Embryonic response to acute anemia varied from complete recovery to malformation and death, depending on the extent of cell death. Malformations occurred only in litters with embryonic deaths. DHA induced low glutathione levels in RBCs, indicating that oxidative stress may be involved in artemisinin toxicity; effects were extremely rapid, with altered RBCs seen as early as GD10.

In establishing the relevance of these findings to humans, one should consider differences in the development of rodents and humans. While yolk sac hematopoiesis occurs similarly in the two species, early placentation and extent of exposure differ. In particular, early hematopoiesis takes only 7 days in rats (during which RBCs expand in a clonal fashion) compared with 6 weeks in humans; thus the susceptible period in relation to the duration of exposure to an artemisinin-based treatment may be substantially different.     

Effects of the benzimidazole anthelmintic drug flubendazole on rat embryos in vitro

Filarial diseases affect millions of people in poverty-stricken areas. In 2011, an investigation of the potential of flubendazole as a safe, highly efficacious, and field-usable macrofilaricidal drug was begun by Drug for Neglected Diseases initiative. As part of the preclinical development program, whole embryo culture was used to investigate the potential embryotoxicity of flubendazole and its metabolites, reduced and hydrolyzed flubendazole. Albendazole was included as a comparator. Flubendazole and albendazole showed similar potency in affecting rat embryonic development in vitro, inducing retardation of growth and dysmorphogenic effects at concentrations ≥0.5 μg/mL. The head, optic and otic systems, branchial arches and posterior body portion were affected. Diffuse areas of cell death were seen in various embryonic districts. The No Observed Effect Level (NOEL) was 0.25 μg/mL for both drugs. Reduced and hydrolyzed flubendazole were less embryotoxic than the parent compound, with NOELs 4-fold and >40-fold higher than that of flubendazole, respectively.     

Embryolethal and teratogenic effects of bromofenofos in rats

Bromofenofos, an organophosphorus anthelmintic, was administered by gavage to rats as a single dose (50 mg/kg) on one of days 6 through 14 of pregnancy. The dams were killed on day 21, and the fetuses were removed, weighed and examined by routine teratological methods. A significant increase in fetal resorptions occurred after administration on days 9 through 13, with a maximum on day 10. Approximately 72% of the implants were resorbed after administration on day 10. Fetal body weights were significantly decreased when dams were treated on day 8 or later. The greatest decrease in fetal body weights was observed on day 10, when the fetuses weighed less than the controls by about 44% on the average. The incidence of fetuses with gross, skeletal and internal malformations was significantly increased on days 8 through 10, on days 8 through 11 and on days 8 and 9, respectively. Although various types of malformations were observed, most of them occurred on day 8, when no significant increase in fetal resorptions did occur. Cleft lip, short tail, brachygnathia, anal atresia, absence of genital tubercle, fused pelvic legs and perineal testicles were seen on day 8 as gross malformations. Skeletal malformations mainly affected the vertebrae and ribs. Major internal malformations on day 8 were hydronephrosis, hydroureter, anophthalmia, cleft palate, agenesis of the bladder and renal agenesis. Anophthalmia and/or microphthalmia were observed on days 8 through 10, with the highest incidence on day 9. To further determine the no-effect levels for embryolethal and teratogenic effects, a single dose of 10, 20, 30 or 40 mg/kg was administered by gavage to rats on days 8 or 10 of pregnancy. The no-effect levels of single oral dose for embryolethal and teratogenic effects were considered to be 40 and 30 mg/kg, respectively.     

葛花对大鼠胚胎致畸作用的观察

目的 观察葛花对SD大鼠胚胎的致畸作用.方法 受孕的雌性SD大鼠随机不分为低、中、高剂量组及对照组,称重并编号.大鼠受孕第7 ～16天,以葛花受试物灌胃,第20天处死,分析大鼠胚胎发育指标与胎仔发育指标,观察胎鼠外观和骨骼有无异常.结果 样品各剂量组孕鼠体重、体重增重、子宫连胎重与对照组比较,差异均无统计学意义（P＞0.05）;样品各剂量组活胎率、死胎率、吸收胎率与对照组比较,差异均无统计学意义（P＞0.05）;样品各剂量组胎仔胎盘重、体重、身长、尾长与对照组比较,差异均无统计学意义（P＞0.05）;样品各剂量组胎仔外观畸形率、内脏畸形率、骨骼畸形率与对照组比较,差异均无统计学意义（P＞0.05）.结论 安全剂量的葛花对大鼠无母体毒性、胚胎毒性和致畸作用,人体每天葛花摄入量7.5g,属安全推荐量.     

In vivo/in vitro study in rat embryos on indium-caused tail malformations

Pathogenesis of indium-caused tail malformations was investigated by in vivo and in vitro experiments. In the in vivo experiment, pregnant Wistar rats received single intravenous administration of indium trichloride at 0.4 mg/kg on day 10 of gestation, and their embryos were examined on days 11, 12 and 13. Embryos in the indium group showed caudal hypoplasia from day 11. Increased apoptosis was observed in their tailbud on day 11. Similar effects were observed in the in vitro experiment, when day 10 rat embryos were cultured in the presence of indium trichloride at 50 microM for 24 h and for further 24 h in the absence of indium. It was considered from these results that caudal hypoplasia probably due to excessive cell loss by increased apoptosis in the tailbud accounted for indium-caused tail malformations in rat fetuses, and that indium-caused embryotoxic effects were direct effects on the conceptus.     

Teratogenic effects of organic extracts from the Pearl River sediments on Xenopus laevis embryos

Toxicity of organic extracts from the Pearl River sediments was investigated with Xenopus laevis embryos. The effects of sediment organic extracts on the mortality, body length and malformation of X. laevis embryos were tested by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The 96-h LC₅₀ values for X. laevis embryos ranged from 62 to 137 g/L (g extracted sediment per L), and the toxicity effect on body length of larvae was not significant under 20 g/L. However, the teratogenic effects produced by sediment organic extracts were diverse, including edema, hypopigmentation, cardiac and ocular malformations, abdomen recurved and curved spine. The percentage of malformations increased with increasing sediment organic extracts, and even reached almost 100% at 10 and 20 g/L in Guangzhou district. A gradient of pollution in the Pearl River sediments was discerned from the teratogenic toxicity. Guangzhou district showed higher teratogenic toxicity compared with Panyu and Nansha districts as a possible consequence of high levels of PAHs, PCBs, OCPs and NP in the sediments. The teratogenic effects of organic extracts from the Pearl River sediments were successfully assessed which indicated the feasibility of teratogenic potential studies of sediments using X. laevis embryos.     

In vivo and in vitro effects of thiuram disulfides and dithiocarbamates on hepatic microsomal drug metabolism in the rat.

The effects of tetramethylthiuram disulfide (TMTDS) and dimethyldithiocarbamate (DMDTC) on hepatic microsomal drug metabolism were studied after in vivo administration to male rats (1 g/kg, po) and after in vitro addition of the compounds to control microsomal suspensions. Results were compared to the effects of the known inhibitor of drug metabolism, disulfiram (DS, tetraethylthiuram disulfide), its reduced metabolite diethyldithiocarbamate (DDTC), and a common metabolite of all four compounds, carbon disulfide. Twenty-four hours after administration of the disulfides (TMTDS and DS) impairment of microsomal aniline hydroxylase and carboxylesterase activities was observed, while cytochrome P-450 and ethylmorphine N-demethylase activity were unchanged. The reduced thiols (DMDTC and DDTC) caused significant decreases in microsomal cytochrome P-450 and impaired all three microsomal enzymes. In vitro addition of all four compounds to control microsomes at a final concentration of 1 mm impaired aniline hydroxylase and carboxylesterase activity. However, only in vitro addition of TMTDS and DS significantly decreased ethylmorphine N-demethylase activity. This effect may be due to the fact that TMTDS and DS bind to cytochrome P-450 producing a type I spectral change and may, therefore, compete with the type I compound, ethylmorphine, for binding sites on cytochrome P-450. Impairment of aniline hydroxylase activity is the most sensitive indicator of an inhibitory effect of all four compounds on microsomal drug metabolism; this action is not dependent on decreases in cytochrome P-450. In vivo impairment of ethylmorphine N-demethylation by DMDTC and DDTC is related to decreases in microsomal cytochrome P-450 produced by these compounds, which may be due, in part, to their decomposition to CS₂ in the gut. The data indicate that industrial or agricultural exposure to compounds such as TMTDS and DMDTC may impair hepatic metabolism, and thereby enhance pharmacological activity of drugs taken by exposed individuals.     

In vitro and ex vivo effects of indobufen on red blood cell deformability

Summary We have studied the effect of indobufen, a cyclo-oxygenase blocking agent which has proved useful in patients with obstructive vascular disease, on red blood cell (RBC) filterability in vitro and in a pilot study ex vivo.The addition of indobufen in vitro to blood samples from 10 healthy volunteers did not significantly modify RBC deformability.We evaluated the ex vivo effect of indobufen (200 mg bd) in 14 patients with obstructive vascular disease. A significant improvement in RBC deformability was noted on the 5th, 14th, and 28th days of treatment, 2 h after the morning dose. Acetylsalicylic acid given to 6 similar patients had no effect suggesting that the positive haemorheological effect of indobufen is probably not linked to its cyclooxygenase blocking effect.     

The interaction of post-weaning housing conditions and prenatal drug effects on behaviour

Offspring of rats given oral doses of imipramine (5 mg/kg) from 14–21 days prior to mating till parturition or vitamin-A (100000 i.u./kg) between days 8 and 10 of gestation were reared, after weaning, in deprived or enriched environments. When tested in both a Henderson-type maze and a swimming maze no behavioural effects due to prenatal drug exposure were observed when the Ss were reared in a deprived environment. When the Ss were reared in an enriched environment the maze performance of those animals which had been exposed to imipramine or vitamin-A was significantly inferior to that of the control animals. The implications of these findings are discussed with respect to environmental interaction with prenatal drug effects on behaviour.     

In vivo and in vitro effect of chelating agents on drug metabolizing enzymes of the rat

Rats given calcium disodium ethylenediaminetetraacetic acid (CaNa₂EDTA), diethylenetriaminepentaacetic acid (DTPA), dimercaprol, p-aminosalicylic acid (PAS), or d-penicillamine (penicillamine) i.p. for 7 successive days showed a significant decrease in the activity of hepatic microsomal benzphetamine N-demethylase. There was no appreciable change in the microsomal cytochrome P-450 concentration. In vitro incubation of the chelating drugs with liver microsomes isolated from rats pre-treated with phenobarbital caused no significant loss of the hemoprotein. The decreased rate of benzphetamine metabolism in microsomal preparations from rats, pretreated with the chelating drugs, may be attributed partly to hepatic depletion of essential trace elements by the chelating drugs.     

In vitro and in vivo investigations on fluoroquinolones; effects of the P-glycoprotein efflux transporter on brain distribution of sparfloxacin.

The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood-brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 microM) on transport of rhodamine-123 (1 microM) was compared with P-glycoprotein inhibitors verapamil (20 microM) and SDZ PSC 833 (2 microM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (-/-) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (-/-) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5+/-2.2 and 1.5+/-0.5%; brain(ECF) concentrations were 5.1+/-0.2 and 26+/-21 microM; and total brain concentrations were 7.2+/-0.3 and 23+/-0.3 microM in wild-type and mdr1a (-/-) mice, respectively. Plasma concentrations were similar (18.4+/-0.7 and 17.9+/-0.5 microM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood-brain barrier.     

In vitro and in vivo assessment of the cardiovascular effects of the cardiotonic drug MDL 19205

Various in vitro and in vivo techniques were used to evaluate the cardiovascular actions of MDL 19205, a new cardiotonic agent. In the anesthetized dog, intravenous administration of MDL 19205 (0.1-1 mg/kg) produced marked increases in cardiac contractile force which were accompanied by small increases in heart rate and minor decreases in blood pressure. These effects were not altered by alpha- or beta-adrenergic receptor blockade, reserpine pretreatment, or bilateral carotid sinus denervation. In isolated cat cardiac tissues, MDL 19205 (10(-5)-10(-3) M) produced a selective inotropic effect relative to isoproterenol and, unlike isoproterenol, was nonarrhythmogenic. Adrenergic beta-receptor or histamine H1-receptor blockade did not modify the inotropic effects of MDL 19205 in guinea pig atria. The vasodilatory effect of MDL 19205 in the canine isolated pump-perfused hindlimb preparation was not significantly attenuated by surgical sympathectomy, alpha- or beta-adrenergic receptor blockade, cholinergic or histaminergic receptor blockade, or indomethacin pretreatment, indicating that MDL 19205 produced direct relaxation of vascular smooth muscle. MDL 19205 was found to be safe and effective when administered acutely in combination with ouabain, hydralazine, nitroglycerin, or furosemide, agents commonly used in the treatment of congestive heart failure. The pharmacological profile revealed by these and other studies suggests that MDL 19205 should be useful in the clinical treatment of congestive heart failure.     

In vivo and in vitro effects of tetrahydroisoquinolines and other alkaloids on rat pituitary function

Several tetrahydroisoquinolines (TIQs) were tested for their in vitro and in vivo capacities to modulate prolactin (PRl) and beta-endorphin (beta-end) secretion by the rat pituitary and for their abilities to displace [3H]spiroperidol and [3H]naloxone binding from pituitary and hypothalamic membranes. Receptor binding studies showed that TIQs could be classified as having (a) higher affinity for opiate receptors (tetrahydropapaverine, papaverine, 6-methylsalolinol, 1-carboxysalsolinol and 3',4'-deoxy-norlaudanosolinecarboxylic acid), (b) higher affinity for the dopamine receptor (salsolinol and 7-methylsalsolinol), or (c) approximately equal affinity for the two binding sites (6,7-dimethylsalsolinol and tetrahydropapaveroline, THP). In freely moving male rats, THP produced a several-fold increase in plasma PRL levels. This effect was not altered by co-administration of naloxone but was attenuated by dopamine. In vitro several TIQs reversed the inhibitory effect of dopamine on PRL secretion by cultured anterior pituitary cells. The order of potencies of the TIQs in this system paralleled their order of potencies in the dopamine receptor assay. THP, the most potent dopamine antagonist, also blocked dopamine-mediated inhibition of beta-endorphin secretion from neurointermediate lobe cells in culture. These data demonstrate that THP and some other TIQs can act as dopamine antagonists in radioreceptor assays, in cell culture and in vivo.     

In vivo effects of benztropine and nomifensine on dopamine and noradrenaline terminals in rat brain

6-Hydroxydopamine (6-OHDA) was given intracerebroventricularly, 200 μg to rats with weight 200–250 g. After 14 days dopamine was measured in the corpus striatum and noradrenaline in the rest of the forebrain. Dopamine decreased to 46% and noradrenaline to 23% of the control values. 4 different drugs, benztropine (1, 5 and 25 mg/kg), nomifensine (10 mg/kg), desipramine (25 mg/kg) and amphetamine (5 mg/kg) were given i.p. 30 min before or 15 min after the 6-OHDA to see if they could prevent the depletion of dopamine and nor-adrenaline. Nomifensine, desipramine and, to a lesser degree, amphetamine prevented the depletion of noradrenaline when given before 6-OHDA, and nomifensine also had some effect when given after 6-OHDA. Nomifensine and amphetamine had an effect on the dopamine content when given both before and after 6-OHDA. Desipramine had no effect on dopamine. The largest dose of benztropine had a small effect on dopamine when given after but not when given before 6-OHDA.The results may in an indirect way give some information about the in vivo action of the drugs on the release and uptake of catecholamines in the brain. Thus it is concluded that amphetamine, desipramine and nomifensine all had an effect on the uptake mechanism in noradrenergic terminals and that in addition nomifensine had a releasing effect on these terminals. Amphetamine and nomifensine but not desipramine both inhibited uptake and provoked the release of dopamine in the dopaminergic terminals. The releasing effect of benztropine on the dopamine terminals may have been too small to have clinical relevance.     

Placental transfer of melamine and its effects on rat dams and fetuses

In 2008, an epidemic of cases of renal failure among Chinese infants, due to melamine contamination of milk, raised international concern. Thus, numerous studies on the metabolism of melamine were broadly undertaken. However, little is known about placental transfer of melamine. In this study, the possibility of placental transfer of melamine and its effects on fetuses and pregnant dams were determined. Melamine was respectively administered at 0, 40 and 400 mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low melamine (LM) and high melamine (HM) groups of pregnant female F344 rats. Rats were sacrificed 30 min after the last gavage. Melamine was not detected in any of the control and placental samples, or in amniotic fluid from the LM group. Plasma and fetal melamine concentrations in the HM group were significantly higher than in the LM group (P < 0.01). Liver enzyme determination revealed no differences among the three groups. However, plasma creatinine, plasma uric acid and blood urea nitrogen concentrations in dams were significantly increased by melamine (P < 0.05). These results show that ingested melamine affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.     

In vivo and in vitro effects of flutamide and diethylstilbestrol on fetal testicular steroidogenesis in the rat

Prenatal testosterone surge is considered crucial for physiological masculinization of male progeny. Disorders in sex steroid hormone balance during the fetal development may interfere with male reproductive health later in life. In this study, we have investigated in utero and in vitro effects of flutamide (FLU) and diethylstilbestrol (DES) on fetal rat testicular steroidogenesis. In utero exposure to FLU 25mg/kg or DES 0.02mg/kg had no obvious effects on ED 19.5 rat testicular testosterone and progesterone production, StAR protein or AR protein expression. However, when ED 19.5 rat testis were cultured for 180min in the presence of 0.1, 1, 10 and 100mg/l of FLU or DES, the highest doses of both compounds were capable of disturbing steroidogenesis. To study the rate of the changes seen in testicular steroidogenesis after 180min, time-series experiments, in which intact testes were cultured with FLU 100mg/l or DES 100mg/l for 30, 60 or 120min, were performed. In vitro time-series experiments revealed that changes in steroidogenesis occur very fast. Experiments with FLU brought further evidence to the hypothesis that ARs have negative autocrine role in developing Leydig cells.     

In vivo effects of nitrogen dioxide and ozone on xenobiotic metabolizing systems of rat lungs

Male Jcl:Wistar rats were exposed continuously to either ozone (O3) or nitrogen dioxide (NO2) for 7 and 14 days to examine the effects of these gases on the xenobiotic metabolizing systems of lung microsomes. Exposure to 0.2 and 0.4 ppm O3 increased NADPH-cytochrome P-450 reductase activity and cytochrome P-450 as well as microsomal protein by the 14th day, whereas NADH-cytochrome b5 reductase was not affected. The most marked increase was observed in cytochrome P-450. In parallel to this increment, the activities of benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase of exposed animals increased significantly on the 7th and 14th days of exposure to 0.2 and 0.4 ppm 03. In contrast, exposure to 1.2 and 4 ppm NO2 decreased cytochrome P-450 on the 7th day. Moreover, the 7-ethoxycoumarin O-deethylase activity in exposed animals decreased to 61% (P less than 0.05) and 74% (P less than 0.001) of control on the 7th and 14th days of exposure to 4 ppm NO2, respectively. This decrease occurred in a dose-dependent manner with exposure to 0.4-4.0 ppm NO2, whilst benzo(a)pyrene hydroxylase activity was not affected. These results show that O3 at low doses induces xenobiotic metabolizing activities in the lung, whereas NO2 reduces this.     

The effect of imipramine administered before and during pregnancy on litter size in the rat

Previous studies have suggested that anti-depressants administered during pregnancy result in an increase in foetal and neonatal morbidity. The present study examined the effects of the anti-depressant imipramine administered before and during pregnancy on litter size in the rat. Imipramine was found to cause a significant reduction in litter size with doses that are used therapeutically.The authors wish to express their thanks to Ciba-Geigy (Australia) Ltd. for supplying imipramine.     

In vivo and in vitro effects of methylmercury on the activities of aminoacyl-tRNA synthetases in rat brain

The activities of six aminoacyl-tRNA synthetase species were determined using enzyme preparations partially purified from the brains of control and methylmercury (MeHg)-treated rats. The activities of Asp-, Leu- and Tyr-tRNA synthetases were significantly reduced in the brains of MeHg-intoxicated rats, whereas those of Lysand Met-tRNA synthetases remained unchanged. In contrast, the activity of His-tRNA synthetase was significantly increased in the symptomatic phase of MeHg intoxication. The activities of these six aminoacyl-tRNA synthetases in the control brains were affected to different extents on the direct addition of MeHg to the assay system in vitro. No positive correlation was observed between the in vivo and in vitro effects of MeHg on the enzyme activities. These results indicate that the aminoacylation of tRNA is one of the actions of MeHg, which leads to inhibition of protein synthesis, and it is suggested that the syntheses of cellular proteins may be modified in different ways by MeHg, depending on their amino acid compositions.This work was supported in part by a grant from the Japanese Environmental Agency.