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Dextran sulphate (DS) stimulates [³H]TdR incorporation in spleen cells of various strains of mice including athymic nude mice. In spleen cells of normal mice an additive effect of DS on the PHA-response was detected. The results suggest that DS is a B-cell mitogen. It seems that the mitogenic activity of polyanions may be related to their effects on antibody formation. [ABSTRACT FROM AUTHOR]

The interaction of the DNA-alkylating model compounds, ethylmethanesulfonate (EMS) and methylnitrosourea (MNU), was studied in pregnant NMRI mice by measuring DNA adduction in vivo. Previously, large-scale dose-response studies on teratogenicity as well as on DNA modification were performed using these substances. In addition, the risk of low doses in mice was estimated by comparative use of several approaches including molecular dosimetry. The risk was further analysed by combination experiments on teratogenesis with EMS and MNU. This paper describes a further approach with regard to an interaction of these compounds: the formation of DNA adducts was determined using a combined treatment regimen of [(14)C]-labelled MNU and EMS. The mutual influence of EMS and MNU on the DNA alkylation rates was found to be moderate. The dramatic increase in the teratogenic outcome following combined treatment found in previous studies was obviously not due to a massive interaction regarding the initial DNA alkylation rates. It may be explained, however, by the concept of toxic equivalency. Teratogenesis Carcinog. Mutagen. 20:27-34, 2000., (Copyright 2000 Wiley-Liss, Inc.)

6-Mercaptopurine riboside (6-MPr) is used as a cytostatic chemotherapeutic. The teratogenic potential in rodents has been well known for several decades. In this study, the teratogenic risk of low doses of 6-MPr in NMRI mice was estimated based on a dose-response study. The effective doses corresponding to the incidences of 5%, 1%, and 0.1% were calculated using the probit and Weibull model. The evaluation was performed on the basis of both the fetus and the litter by evaluating the variable all gross structural abnormalities. From these experiments, benchmark doses were obtained which were used in low-dose risk assessment to define a reference dose. Depending on the biometrical model and the statistical unit used, values between 1.9 and 5.2 mg/kg (benchmark ED1) and 3.8 and 6.7 mg/kg (benchmark ED5) were obtained. These values were compared to the no observed adverse effect level (NOAEL) which was determined experimentally. The NOAEL was found to be 5 mg/kg, which is quite similar to the ED5 benchmark doses.

In previous studies the direct-acting alkylating model compounds methylnitrosourea (MNU) and ethylmethanesulfonate (EMS) were investigated with regard to dose-response of teratogenicity as well as DNA adduct formation in mice. In this study the teratogenic effects induced by combined treatment with these substances were analyzed using doses which, following single treatment with either substance, were around the threshold level, i.e., no adverse effect level (NOAEL) and lowest observed adverse effect level (LAOEL). Combined treatment of LAOELs resulted in a threshold-like response, while the combination of the NOAEL of one substance with the LAOEL of the other increased the response rate dramatically to nearly 100%. This phenomenon was further evaluated using biometrical methods. The dose-response surface as well as isobolograms were calculated in order to describe the combination effect. In addition, a dose-response model was fitted to the data. In conclusion, the initially surprising high combination effect revealed to be not so extraordinary when considering the steepness of the dose-response relationships of the single substances.

Analogously to an earlier study using methylnitrosourea (MNU) the prenatal-toxic risk of low doses of ethylmethanesulfonate (EMS) was estimated using different procedures, comparatively. First, the risk of low doses was estimated using linear extrapolation to zero. When using the variable "all gross structural abnormalities" the lowest effective dose in the experiment was 150 mg/kg body wt (5.6% incidence), the additional risk over background was calculated to be 5.0%, and the hypothetical incidence 0.1% was associated with the dose 3 mg/kg EMS. When evaluating "gross structural limb abnormalities," which are not observed in controls, the dose associated with the hypothetical incidence 0.1% was 17.4 mg/kg EMS. Furthermore, derived from a dose-response study of teratogenicity extrapolation to the possible risk of low doses was performed using nonlinear mathematical models. In this case, the results obtained are dependent on the dose response variable as well as from the statistical approach which was chosen. As an example, the values obtained from one evaluation are given: all gross structural abnormalities, Weibull transformation, jackknife approach: ED0.1% = 72 mg/kg EMS. For comparison a "virtually safe dose" was calculated by use of the no-observed-adverse-effect-level (NOAEL) risk factor approach. The NOAEL under our experimental conditions was 100 mg per kg body wt. By using an arbitrarily chosen risk factor of 100 a "safe dose" of 1 mg EMS per kg body wt was obtained. In addition, molecular dosimetry of the DNA adduct rate of O6-ethylguanine in the 11-day-old embryos was used. Based on the assumption that a linear correlation exists between this specific adduct rate and the incidence of teratogenic effects, the hypothetical incidence of 0.1% was associated with a dose of 99 mg/kg EMS. This value is quite similar to that obtained by extrapolation using probit analysis which is in contrast to the results obtained with MNU.

In previous studies using methylating agents a correlation was found between the initial DNA adduct rate (O6-methylguanine) in the embryo and the teratogenic efficiency. This was shown by measuring DNA adduct rates in the teratogenic dose range which exhibited similar adduct rates at the equivalent teratogenic dose levels. A similar approach was performed using the ethylating agent ethylmethanesulfonate (EMS). In the teratogenic dose range (150-250 mg/kg bw) the adduct rates of O6-ethylguanine were similar compared to those of O6-methylguanine which were obtained with methylating agents. We conclude that a correlation between teratogenicity and adduct rate (O6-alkylguanine) exists for both methylating and ethylating agents. Furthermore, DNA adduct formation following doses at and below the no-observed-adverse-effect-level (NOAEL) of teratogenicity was determined. The lowest experimental dose was 45 mg/kg EMS. Substantial DNA adduct rates in the embryos were found. These data will be used for molecular dosimetry in a risk assessment of low doses.

Prenatal-toxic risk estimation for the alkylating model compound methylnitrosourea (MNU) was performed using different procedures. Risk of low doses was estimated using linear extrapolation to zero (estimated ED0.1%: 0.1 mg/kg body wt MNU) as well as extrapolation by probit analysis based on a dose-response study (estimated ED0.1%: 1.6 mg/kg body wt). Furthermore, a "virtually safe dose" was established by means of the NOAEL risk factor approach (e.g., factor 30:0.03 mg MNU per kg body wt). In previous studies in murine embryos using MNU, we combined dose-response data and DNA adduct rate measurements and deduced that O6-methylguanine is a suitable variable for molecular dosimetry. In a tentative approach, we estimated the teratogenic risk of low doses based on the adduct rates of O6-methylguanine in the DNA of the embryos. It is concluded that in the case of steep dose-response relationships, which are typical for the majority of teratogenic effects, the NOAEL risk factor approach is more conservative than extrapolation based on probit analysis. Risk estimation using dosimetry with this model compound yields estimated incidences similar to linear extrapolation.

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 micrograms/kg body wt; maintenance dose: 5 micrograms/kg body wt, once weekly) and TCDD-75 (initial dose: 75 micrograms/kg body wt; maintenance dose: 15 micrograms/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25 = 5.1%, controls = 2.6%), and incompletely ossified ossa zygomatica (TCDD-25 = 1.8%, controls = 0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25 = 1.3%, controls = 0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls = 0.6%). There was no difference in postnatal mortality (TCDD-25 = 1.3%, controls = 1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.

Formation of DNA adducts in 11-day-old mouse embryos was studied by measuring the initial alkylation rates of the methylated purine bases 7-methylguanine, O6-methylguanine, and 3-methyladenine. In the first part of the studies the adduct rates were measured in the teratogenic dose range (ED10-ED90, 2.7-5.6 mg/kg). These results were compared with similar data obtained from studies with ethylmethanesulfonate and acetoxymethyl-methylnitrosamine. For the three investigated substances a correlation was found between the initial adduct rate of O6-alkylguanine in the DNA of the embryos and the teratogenic potency. In the second part of the study the rate of adduct formation was measured in the sub-teratogenic dose range. These data will be used for molecular dosimetry in a risk assessment of low doses.

It has been reported that fractionated doses of 13-cis-retinoic acid are disproportionately more embryotoxic in pregnant mice than is the same dose given in a single bolus. Here, we examined limited pharmacokinetic profiles of a single (100 mg/kg dose given to NMRI mice on day 11 of gestation) versus multiple (3 x 100 mg/kg, 4 h apart) doses in an effort to assess the relative contribution to teratogenicity made by the drug and/or its metabolites. The major plasma metabolite of 13-cis-retinoic acid in the mouse was 13-cis-retinoyl-beta-glucuronide, followed by the 4-oxo metabolites and all-trans-retinoic acid. Transfer to the mouse embryo was very efficient for all-trans-retinoic acid, whereas, it was tenfold less efficient for 13-cis-retinoic acid and 100-fold less efficient for 13-cis-retinoyl-beta-glucuronide. The isomer all-trans-retinoic acid was found in the placenta at concentrations two- to three-fold higher than in the plasma, suggesting placental accumulation as well as placental cis/trans isomerization. Since 13-cis-retinoyl-beta-glucuronide and 13-cis- and all-trans-retinoic acid were detected in the embryo after this multiple dosing schedule, any of the three or their combinations may have been involved in the induction of malformations, but all-trans-retinoic acid, a well-known potent teratogen detected at concentrations of between 590 and 80 ng/g for 10 critical hours during gestation, could have been the major component.

NMRI mice were treated on day 11 (day 0 = plug day) of gestation with a single oral dose of 100 mg/kg of either all-trans-etretin (acitretin) or 13-cis-etretin. For teratology studies mice were sacrificed on day 18 of gestation, and the fetuses were examined for malformations. For pharmacokinetic studies, groups of 5 mice were sacrificed after 5, 10, and 30 min and 1, 2, 4, and 8 h. The concentrations of retinoids in maternal plasma and in embryos were determined by a newly developed HPLC gradient method. All-trans-etretin induced malformations in 94% of the fetuses, mainly in fore and hind limbs and cleft palate. 13-cis-etretin did not show any teratogenic or embryo-lethal effects at the dose level used. These findings could be explained by a transplacental pharmacokinetic study. The maximum peak level and also the AUC (area under the concentration-time curve) value of all-trans-etretin in the embryos was 7-8 times higher than corresponding values for 13-cis-etretin, probably due to extensive transport of the trans-isomer and limited transport of the cis-isomer from maternal plasma to the embryos. The concentration quotient between embryo and the maternal plasma was between 0.43 and 1.10 for all-trans-etretin, and only 0.16-0.31 for 13-cis-etretin over the time period studied. An in vivo isomerization of the compounds was also observed which was more extensive for 13-cis-etretin than for all-trans-etretin. Our results indicate that the low teratogenic potency of 13-cis-etretin is due to a limited placental transfer of this compound; on the other hand, the potent teratogen all-trans-etretin is rapidly and extensively transferred to the embryo.

The alkylating agent acetoxymethyl-methylnitrosamine (DMN-OAc) triggers preferential left-sided paw defects in mice following IP administration on either day 11 or 12 of pregnancy. Predominantly, ectrodactyly and hypoplasia of the left paws were found. In an organ culture system, using limb buds of 11-day-old mouse embryos, differentiation is severely impaired following addition of 2 microM DMN-OAc to the culture medium. Left and right limbs are equally affected. In contrast, when DMN-OAc is administered in vivo to the dams with subsequent culturing of the limb buds, growth and differentiation of the left limb buds is more affected when compared to the right. Furthermore, DNA alkylation experiments were performed: in vitro, following addition of (14C)-DMN-OAc (2.3 microM) to the culture medium, the DNA alkylation rate of the limb bud DNA is determined. In vivo, following IP administration of 10 mg/kg DMN-OAc to the dams on day 11 of pregnancy, the extent of DNA alkylation of whole-embryo DNA is similar. However, the DNA alkylation rate of separately pooled left and right limb buds exhibits a two-fold difference according to the different teratogenic susceptibility. The results obtained with both in vivo and in vitro systems are consistent with the thesis that a certain amount of DNA alkylation in the tissue of the embryos is the initial step of alkylating agent-induced teratogenicity.

Following three s.c. injections of acyclovir (100 mg acyclovir/kg) into rats on day 10 of pregnancy 19 litters were evaluated on day 21 of gestation and the effects were compared to the results obtained from controls (nine litters) which received the vehicle (0.1 N NaOH) only. The following results were obtained (treated group versus control group): 1) Implantations/litter: 11.2 +/- 1.3 versus 10.2 +/- 1.1; 2) resorptions/implantations: 27.7% versus 2.2%; 3) number of viable fetuses evaluated: 154 versus 90; 4) fetuses with anomalies of the skull: 78% versus 12%; 5) fetuses with anomalies of the vertebral column: 38% versus 13%; 6) gross-structural anomalies predominantly affected the skull and tail. The most frequently registered defects were: os tympanicum (smaller): 23%, os tympanicum (missing): 23%; missing tail: 7%; protruding tongue (15%); none of these defects were seen in the control fetuses. Postnatally we observed a high mortality rate among the offspring. From a total of 85 newborn (nine litters) we obtained 73 viable offspring (9.1 +/- 3.4); 81% of them had tail alterations. In the control group of eight litters (9.4 +/- 2.3) no tail alterations occurred. On day 21 postnatally 40 viable offspring were alive (mortality rate: 38.8%). Nearly all of these animals had visible alterations at multiple sites of their bodies; most frequently observed were: tail impairment, closed eyes, dragging hind-limbs, and urogenital alterations (e.g. testicular atrophy). These studies how for the first time that prenatal treatment with acyclovir induces gross-structural defects which persist postnatally.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte reactivity in vitro was studied in patients with Hodgkin's disease, chronic lymphocytic leukaemia and lymphosarcoma. The responding capacity to phytohaemagglutinin (PHA) was markedly depressed and delayed in all three groups of patients compared with the PHA response observed in lymphocyte cultures from normal individuals. In one-way mixed lymphocyte culture experiments a significant decrease in responding capacity of the patients' lymphocytes to lymphocytes from normal donors could be demonstrated. In contrast, the stimulatory capacity of the patients' lymphocytes was found to be intact, or only slightly reduced.

Pregnant mice received 10 or 100 mg retinol/kg body wt. by gavage on day 11 of gestation (plug day = day 0). One group of animals was used for a pharmacokinetic study. At various times after dosing, plasma and tissue samples were collected and analyzed by HPLC for retinyl esters, retinol, 13-cis- and all-trans-retinoic acid and 13-cis-4-oxo and all-trans-4-oxoretinoic acid. In the other group the fetuses were removed on day 18 and examined for malformations. After 10 mg/kg retinol, no teratogenic effect was observed. The pharmacokinetic investigation revealed a moderate increase of retinyl esters, retinol and all-trans-retinoic acid in plasma, embryonic tissue, placenta, yolk sac membranes and extraembryonic fluid. A high incidence of severe fetal malformations occurred after 100 mg/kg retinol. These malformations included limb defects (81% of fetuses) and cleft palate (55% of fetuses) which are characteristically found after administration of a single teratogenic dose of an active retinoid on day 11 of gestation. The concentration-time profile of retinoids after 100 mg/kg on day 11 showed a pronounced increase of retinyl esters and retinol in all compartments including the embryo and a massive generation of the polar metabolites all-trans-retinoic acid and all-trans-4-oxoretinoic acid. These polar metabolites were found in the embryo with peak concentrations of 327 +/- 115 and 143 +/- 20.7 ng/g (mean +/- SE) wet tissue, respectively. It is likely that all-trans-retinoic acid and all-trans-4-oxoretinoic acid, both well-known teratogens, largely contributed to the teratogenic outcome. The in-vivo oxidation of retinol may be an important factor in the teratogenic activity of high doses of vitamin A.

Pregnant rats were treated during organogenesis with s.c. injections of acyclovir and the embryos were evaluated on day 11.5 of gestation (crown-rump length, somites, protein content, score, abnormalities, histological examination). After eight injections of 50 mg/kg body wt on days 9, 10, and 11 of pregnancy a reduction of the crown-rump length was noticed. After 100 mg/kg this effect was more pronounced. With two or three applications of this dose on day 10 specific embryonic abnormalities were visible: the shape of the head was abnormal, the width of the skull had decreased resembling a beak-like visceral cranium. With a single administration of 200 mg/kg on day 10 we found a similar but slightly more pronounced outcome. A drastic change of all variables was obtained after eight injections of 100 mg/kg on days 9, 10, and 11. Comparatively we measured maternal plasma concentrations of acyclovir 1 h after the administration of 50, 100 or 200 mg/kg body wt. After an injection of 50 mg/kg on days 9, 10, and 11 of gestation (three injections/day) the plasma levels ranged from 19.1 to 40.0 mg/l (1 mg/l = 4.44 microM). No cumulation was observed. In contrast, a cumulative effect was detected following a dose of 100 mg/kg. After the first injection of this dose a mean value (+/- SD) of 60.3 +/- 14.7 mg/l (n = 16) was obtained. In this case a third injection increased the mean plasma level to 124.6 +/- 16.6 mg/l (n = 5). Further injections, however, led to decreasing levels. One hour after administration of 200 mg/kg body wt acyclovir levels ranged from 120.0 to 163.9 mg/l. We conclude that acyclovir, at doses leading to plasma concentrations well above the therapeutic level in the dam, interferes with the embryonic development in the rat. Acyclovir induces typical gross structural abnormalities which have been first observed using a whole embryo culture system.

The teratogenic potency of the directly acting alkylating agent methylnitrosourea (MNU) was analysed in mice. Skeletal abnormalities were evaluated after treatment on either day 11 or 12 of pregnancy. Ectrodactyly was the predominant effect after treatment on day 11. Treatment on day 12 triggered especially double-sided microdactyly (method of analysis: measuring digit lengths). Litter variabilities were analysed using a new biometrical procedure. Using probit analysis, dose-response curves were computed from the experimental data obtained and the effective doses were calculated and compared with maternal toxicity. Low dose extrapolation was performed by use of various mathematical models which yielded very similar ED1/100 and ED1/1000 values.