Cylindrospermopsin toxicity in mice following a 90-d oral exposure.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

A critical review of the postulated role of the non-essential amino acid, ß-N-methylamino-L-alanine, in neurodegenerative disease in humans.

The compound BMAA (ß-N-methylamino-L-alanine) has been postulated to play a significant role in four serious neurological human diseases: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, Parkinsonism, and dementia that occur globally. ALS/PDC with symptoms of all three diseases first came to the attention of the scientific community during and after World War II. It was initially associated with cycad flour used for food because BMAA is a product of symbiotic cycad root-dwelling cyanobacteria. Human consumption of flying foxes that fed on cycad seeds was later suggested as a source of BMAA on Guam and a cause of ALS/PDC. Subsequently, the hypothesis was expanded to include a causative role for BMAA in other neurodegenerative diseases including Alzheimer's disease (AD) through exposures attributed to proximity to freshwaters and/or consumption of seafood due to its purported production by most species of cyanobacteria. The hypothesis that BMAA is the critical factor in the genesis of these neurodegenerative diseases received considerable attention in the medical, scientific, and public arenas. This review examines the history of ALS/PDC and the BMAA-human disease hypotheses; similarities and differences between ALS/PDC and the other diseases with similar symptomologies; the relationship of ALS/PDC to other similar diseases, studies of BMAA-mediated effects in lab animals, inconsistencies and data gaps in the hypothesis; and other compounds and agents that were suggested as the cause of ALS/PDC on Guam. The review concludes that the hypothesis of a causal BMAA neurodegenerative disease relationship is not supported by existing data.

The course of toxicity in the pregnant mouse after exposure to the cyanobacterial toxin cylindrospermopsin: clinical effects, serum chemistries, hematology, and histopathology.

Cylindrospermopsin (CYN) is a toxin produced by a variety of fresh-water cyanobacterial species worldwide and induces significant adverse effects in both livestock and humans. This study investigated the course of CYN-induced toxicity in pregnant mice exposed daily during either the period of major organogenesis (gestation days [GD] 8-12) or fetal growth (GD13-17). Endpoints include clinical signs of toxicity, serum analyses to evaluate hepatic and renal function, histopathology of liver and kidney, and hematology. Study animals were administered 50 µg/kg CYN once daily by ip route and euthanized 24 h after 1, 2, 3, 4, or 5 consecutive doses, or 6 or 13 d after the dosing period. The course of the CYN-induced effects was determined at all euthanasia times for the endpoints just outlined. Results indicated that CYN is a toxin, producing lethality in dams during the early part of gestation, significant weight loss, and bleeding in the gastrointestinal tract, tail tip, and peri-orbital tissues. Effects also included alterations in serum markers for liver function, histopathological changes in liver and kidney tissues, electrolyte abnormalities, leukocytosis, and posttreatment thrombocytopenia and reticulocytosis. The onset of symptoms was rapid, producing reductions in weight gain in GD8-12 animals, bleeding in the vaginal area in GD13-17 animals, and significant increases in sorbitol dehydrogenase (SDH) in both groups after a single dose. Although the GD8-12 dams displayed a 50% lethality, in GD13-17 animals only a single death occurred. Alterations seen in hepatic and renal function or histopathology do not appear to be of sufficient severity to produce death. Evidence indicates that bleeding may play a critical role in the onset of symptoms and eventually, in the observed lethality.

Susceptibility of adult and senescent Brown Norway rats to repeated ozone exposure: an assessment of behavior, serum biochemistry and cardiopulmonary function.

Ozone (O3) is a pervasive air pollutant that produces pulmonary and cardiovascular dysfunction and possible neurological dysfunction. Young and old individuals are recognized as being susceptible to O3; however, remarkably little is known about susceptibility with senescence. This study explored the pulmonary, cardiovascular and neurological effects of O3 exposure in adult (4 m) and senescent (20 m) Brown Norway rats exposed to 0 or 0.8 ppm O3 for 6 h, 1 d/week, for 17 weeks. Ventilatory function was assessed 1 and 7 d after each exposure (Buxco). Heart rate, blood pressure (tail cuff) and motor activity were measured biweekly. Blood, aorta and bronchoalveolar lavage fluid (BALF) were analyzed 24 h after the last exposure for pulmonary inflammation, serum biomarkers and aorta mRNA markers of vascular disease. Measures of normal ventilatory function declined following each O3 exposure in both adult and senescent rats, however, senescent rats took weeks to exhibit a decline. Evidence for residual respiratory effects of O3 7 d after exposure in both age groups was observed. O3 had no effect on either heart rate or blood pressure, but decreased motor activity in both age groups. BALF indicated mild neutrophilic inflammation and protein leakage in adults. Age affected 17/58 serum analytes, O3 affected 6/58; 2/58 showed an age-O3 interaction. Leptin, adiponectin, lipocalin and insulin were increased in senescent rats. Overall, adult rats exhibited more immediate effects of episodic O3 than senescent rats. Residual effects were, however, obtained in both ages of rat, especially for ventilatory endpoints.

Toxicity and recovery in the pregnant mouse after gestational exposure to the cyanobacterial toxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post-dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8-12 induced significantly more lethality than GD13-17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8-12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13-17 group. Gene expression changes persisted up to 2 weeks post-dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter-animal variability within the treated groups.

Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture.

The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic acid are developmentally toxic in mouse whole embryo culture. Human exposure to these contaminants in drinking water would involve simultaneous exposure to all three HAs. This study explores the question of developmental toxicity interactions between these compounds. Gestational day (GD) 9.5 rat embryos were exposed to various concentrations of the three HAs (singly or in combination) for 48 h and then evaluated for dysmorphology. The embryonic effects from exposure to the single compounds and mixtures were evaluated using developmental score (DEVSC) as the parameter of comparison. Concentrations of individual compounds and mixtures were chosen (based on a dose-additivity model) which were predicted to produce scores 10 or 20% lower than control levels. Evaluations were performed on all possible combinations of the three HAs. The HAs were dysmorphogenic and resulted in primarily rotation and heart defects and to a lesser extent prosencephalic, visceral arch, and eye defects. The percent anomalies in the rat were comparable to those previously published for the mouse at comparable toxicant concentration. There was a low incidence of neural tube defects in the rat following exposure to the HAs. The rat neural tube appeared less sensitive to the HAs than did the mouse resulting in a higher rate of neural tube dysmorphology in the mouse. Following exposures to BCA and DBA, alone and in combination, there was a significant incidence of delayed embryonic caudal development with apparent normal development anterior to the second visceral arch. The developmental scores for embryos exposed to combinations of the three compounds, when compared to scores for embryos exposed to the single compounds, indicated that the dose-additivity model adequately predicted the observed toxicity and that the developmental toxicity of these water disinfection by-products appears to be additive in whole embryo culture (WEC).

Gametic embryos of maize as a target for biolistic transformation: comparison to immature zygotic embryos.

The aim of the present study was to determine the suitability of maize gametic embryos of three ETH genotypes as a target for biolistic transformation. We studied parameters considered essential for a successful transformation, such as the frequency of secondary embryo formation, their regeneration ability and the transient transgene expression. Transformable zygotic embryos of one of the ETH genotypes were used as positive control. Our results indicate that gametic embryos can potentially be transformed by particle bombardment, since they responded positively to all the studied parameters, although with lower efficiencies than the zygotic embryos. In particular, differences were found in the rate of secondary embryogenesis and the density of transformed cells.

Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-alpha.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are correlated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor-alpha (TGF-alpha), and altered epithelial cell proliferation and differentiation. This research examined the role of these growth factors in TCDD-induced teratogenicity by using wild type (WT) and knockout (-/-) mice that do not express EGF, TGF-alpha, or both EGF and TGF-alpha. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 microg/kg, 5 ml/kg. On GD 17.5, the maternal parameters evaluated included body weight, body weight gain, liver weight (absolute and adjusted for body weight). The number of implantations, live and dead fetuses, early or late resorptions, the proportion of males, fetal body weight, fetal absolute and relative liver weight, placenta weight, incidence of cleft palate, and the severity and incidence of hydronephrosis were recorded. TCDD did not affect maternal weight gain, fetal weight, or survival, but maternal and fetal liver weights and liver-to-body weight ratios were increased in all genotypes. The WT and TGF-alpha (-/-), but not the EGF (-/-) and EGF + TGF-alpha (-/-) fetuses, developed cleft palate after exposure to 24 microg TCDD/kg. Hydronephrosis was induced by TCDD in all genotypes, with the incidence in EGF + TGF-alpha (-/-) fetuses comparable to that of the WT. The incidence and severity of this defect was substantially increased in EGF (-/-) and TGF-alpha (-/-). In conclusion, this study demonstrated that expression of EGF influences the induction of cleft palate by TCDD. Also, EGF and TGF-alpha are not required for the induction of hydronephrosis, but when either is absent the response of the fetal urinary tract to TCDD is enhanced.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse.

The aryl hydrocarbon receptor (AHR) is a transcriptional regulatory protein that binds to upstream DNA response elements of target genes. Activation of the AHR by binding of ligands such as polyhalogenated dioxins, furans, and PCBs is associated with a wide range of adverse biological outcomes, including cancer, immune deficiencies, embryo/fetotoxicity, and reproductive toxicity. Investigations of the diverse biological responses mediated by the AHR led to production of a transgenic mouse in which the gene coding for the AhR was inactivated. AHR-deficient mice were fertile and at maturity exhibited immune system impairment and hepatic fibrosis. Our laboratory received several of these homozygous knockout (-/-) mice and mated them with wild-type (+/+) C57BL/6N mice to generate large numbers of heterozygotes (+/-). The -/- males were then mated with a total of 45 heterozygous +/- females. Offspring of these matings were genotyped and mated in all genotypic combinations. Although male and female -/- adults were fertile, the -/- females had difficulty maintaining conceptuses during pregnancy, surviving pregnancy and lactation, and rearing pups to weaning. Only 46% of the 39 pregnant -/- females successfully raised pups to weaning. The -/- pups showed poor survival during lactation (average death rate per litter was 16%) and after weaning (26.5% of the 230 weaned -/- pups died within 2 weeks). Only 39% of the implantations in uteri of -/- dams resulted in offspring surviving to Postnatal Day 45. Across all litters the sex ratios and genotypic frequencies were comparable to expected values. Reproductive success was adversely affected in Ahr-null females and conceptuses. Additional study is needed to reveal the etiology of these effects.

RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.

Improved formation of regenerable callus in isolated microspore culture of maize: impact of carbohydrates, plating density and time of transfer.

Pure fractions of maize (Zea mays L.) microspores at various densities were exposed to defined media containing different concentrations of maltose and sucrose. In general, lower carbohydrate concentrations (60, 90 g/l) yielded higher frequencies of embryo-like structures than a high concentration (120 g/l). Optimum cell density seemed to depend on the genotype, but densities above 80,000 microspores/ml led to reduced embryogenesis in all genotypes tested. Direct comparison of maltose and sucrose as carbohydrate source in the induction medium clearly demonstrated the superiority of maltose with regard to the regeneration frequency. For two out of three genotypes tested, maltose also enhanced the formation of embryo-like structures. The time of embryo transfer to callus induction media had a significant effect on regeneration frequency.

Effects of combinations of methanol and formic acid on rat embryos in culture.

Acute human exposure to methanol (MeOH) results in elevated serum concentrations of both MeOH and formic acid. In order to better assess the risk of adverse developmental effects of MeOH exposure in humans, the effects of the combination of formate and MeOH, in addition to the individual toxicity profiles for MeOH and formate, need to be established. Gestational day 9 rat embryos were exposed to various concentrations of MeOH and formate in whole embryo culture (WEC) for 48 hr and the degree of embryotoxicity was evaluated using developmental score (DEVSC) as the parameter of comparison across exposure combinations. After establishing embryo toxicity of the individual compounds in previous studies, concentrations of MeOH and formate were chosen which would produce similar DEVSCs, and isoboles were plotted joining the equivalently toxic doses. These mixtures would be expected to have similar toxicity to the MeOH or formate concentrations according to a dose-addition model. The responses of embryos to the selected concentrations along each isobole were measured and tested for linearity to determine the nature of any interaction between the two agents. The concentrations of MeOH and formate used separately and in combination ranged from 0 to 8.75 mg/ml MeOH and 0 to 1.51 mg/ml formate. Increasing concentrations of either MeOH or formate resulted in significant decreases in DEVSC. Exposure to combinations of MeOH and formate had less effect on DEVSC than would be expected based on simple toxicity additivity. This observation was also true for embryonic crown-rump length, head length, and somite number. These results suggest that the embryotoxicity observed following low level exposure to MeOH is mechanistically different from that observed following exposure to formate.

Effects of boric acid on axial skeletal development in rats.

Prenatal exposure to elevated levels of boric acid (BA) causes reduced incidences of supernumerary ribs and shortening/absence of the 13th rib in multiple laboratory species. To explore this further, Sprague-Dawley rats received 500 mg/kg b.i.d. on gestation days (gd) 5-9, 6-9, 6-10, or on single days between gd 6 and 11 (plug day = gd 0); gd-21 fetuses were stained for skeletal examination. Following multiday exposures, malformations of the axial skeleton involved the head, sternum, ribs, and vertebrae. Shortening/absence of the 13th rib was seen particularly in the gd 5-9 and 6-10 exposure groups. Although most groups exposed on single days were generally unaffected, about 90% of the gd-9 exposed fetuses had only six cervical vertebrae; the deficient region was usually C3-C5. In contrast, gd-10 treatment caused agenesis of a thoracic/lumbar vertebra in over 60% of the fetuses; the deficient region was usually T11. For 13-ribbed fetuses, the length of rib 13 was shortened compared to controls. Postnatal assessment suggested increased mortality for gd-10 exposed pups. Embryos in culture showed reduced development when exposed to BA for 48 h. These findings demonstrate the critical periods for axial development in the rat and provide an experimental model for the study of homeotic shifts.

Improved production of doubled haploids by colchicine application to wheat (Triticum aestivum L.) anther culture.

The aim of this study was to optimize the in vitro chromosome-doubling procedure in wheat anther culture. Colchicine, at concentrations of 100-5000 mg/l, was added to the induction medium for 1-5 days. Beneficial effects were obtained with concentrations of 100 and 1000 mg/l colchicine. With time, significant reductions in embryo-like structures as well as higher doubling indices were found. Similar results were obtained with the high- and low-responding genotypes. Colchicine (100 mg/l), added 5 and 20 days after inoculation for 1 and 3 days increased the induction response, but this value was reduced when colchicine was added 10 or 15 days after inoculation. The doubling effect was similar to the control, except for a significant increase with the 3-day application 20 days after inoculation. The highest success index was reached when colchicine was added to the culture medium after 20 days.

Comparative effects of haloacetic acids in whole embryo culture.

A major class of disinfection by-products in drinking water are the haloacetic acids. Both dichloro- and trichloroacetic acids are teratogenic when administered to rats throughout organogenesis. However, there is little information regarding the developmental toxicity of other haloacetic acids. Therefore, 3-6 somite staged CD-1 mouse embryos were exposed to acetic acid (AA) or mono- (M), di- (D), and tri- (T) substituted fluoro- (F), chloro- (C), or bromo- (B) acetic acids in whole embryo culture in order to evaluate the effects of these agents on development. A 24 hour exposure to the haloacetic acids produced dysmorphogenesis. Effects on neural tube development ranged from prosencephalic hypoplasia to non-closure defects throughout the cranial region. Exposure to the haloacetic acids affected optic development, produced malpositioned and/or hypoplastic pharyngeal arches, and resulted in perturbation of heart development. In order to determine the relative toxicities of these agents, benchmark concentrations were calculated as the lower 95% confidence interval of the concentration that produced a 5% increase in neural tube defects. The benchmark concentrations occurred over a wide range with DFA (5912.6 microM) and MBA (2.7 microM) at the extremes. Using the benchmark concentrations to compare the chemicals gives a ranking of the agents in order of increasing potency as: DFA < TFA < DCA < AA < TBA < or = TCA < DBA < MCA < MBA. TCA and DCA have demonstrated ability to disrupt development in vivo but were among the least potent haloacetic acids in vitro. Because of the potential for widespread exposure to haloacetic acids in drinking water and the incomplete toxicity profile of these chemicals, further work on their developmental effects is warranted.

A simulation study of the influence of study design on the estimation of benchmark doses for developmental toxicity.

The benchmark dose (BMD)4 approach is emerging as replacement to determination of the No Observed Adverse Effect Level (NOAEL) in noncancer risk assessment. This possibility raises the issue as to whether current study designs for endpoints such as developmental toxicity, optimized for detecting pair wise comparisons, could be improved for the purpose of calculating BMDs. In this paper, we examine various aspects of study design (number of dose groups, dose spacing, dose placement, and sample size per dose group) on BMDs for two endpoints of developmental toxicity (the incidence of abnormalities and of reduced fetal weight). Design performance was judged by the mean-squared error (reflective of the variance and bias) of the maximum likelihood estimate (MLE) from the log-logistic model of the 5% added risk level (the likely target risk for a benchmark calculation), as well as by the length of its 95% confidence interval (the lower value of which is the (BMD). We found that of the designs evaluated, the best results were obtained when two dose levels had response rates above the background level, one of which was near the ED05, were present. This situation is more likely to occur with more, rather than fewer dose levels per experiment. In this instance, there was virtually no advantage in increasing the sample size from 10 to 20 litters per dose group. If neither of the two dose groups with response rates above the background level was near the ED05, satisfactory results were also obtained, but the BMDs tended to be more conservative (i.e., lower). If only one dose level with a response rate above the background level was present, and it was near the ED05, reasonable results for the MLE and BMD were obtained, but here we observed benefits of larger dose group sizes. The poorest results were obtained when only a single group with an elevated response rate was present, and the response rate was much greater than the ED05. The results indicate that while the benchmark dose approach is readily applicable to the standard study designs and generally observed dose-responses in developmental assays, some minor design modifications would increase the accuracy and precision of the BMD.

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.).

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5-1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1-7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1-3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal.

Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.

Gardnerella vaginalis infection and sexual contact in female children.

Gardnerella vaginalis (GV) infection has been reported as being acquired via sexual contact in adults and as an indicator of sexual contact in female children (DeJong, 1985). The purpose of this study was to determine if GV infection was more commonly found in 191 female children who gave a history of sexual contact and/or were infected with Neisseria gonorrhoeae (GC) or Chlamydia trachomatis (CT) (Group 1), compared with 144 female children evaluated for possible sexual abuse and found to have no such history or infection with GC or CT (Group 2), or 31 female children (friends of the authors) without such a history or GC or CT infection (Group 3). Vaginal GV was found in 5.3% of Group 1, 4.9% of Group 2 and 6.4% of Group 3 (p > .05). Also, vaginal GV infection was not related to the type of sexual contact or race, but did increase with age in white female children. Because vaginal GV infection is not more commonly found in children with a history of sexual contact than those without such a history, the finding of GV in a vaginal culture in an individual case would not be a reliable marker of sexual contact. Routine culturing for GV is not recommended as part of a sexual abuse workup.