bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of human spermatozoa for Y chromosomal nondisjunction
Authors: Kapp R. W. , Jacobson C. B.
Published: February 2006

The YFF sperm assay, which is a quantification of the incidence of sperm with two fluorescent bodies (YFF = two fluorescent bodies), was performed to measure Y chromosomal nondisjunction. Three categories of human sub‐ jects were analyzed: (1) nonexposed, (2) exposed to antineoplastic agents ‐ ie, chemo‐ and radiation therapy, and (3) dibromochloropropane (DBCP)‐exposed. The nonexposed individuals demonstrated a relatively constant mean YFF value of 1.3%, which was consistent with historical controls in this laboratory and with the results of other investigators. Further, a one‐way analysis of variance among the means of the control samples revealed no statistical differ‐ ences either between these men or within each man's samples. The individuals exposed to antineoplastic agents showed a three‐ to four‐fold increase in the incidence of YFF sperm three to six weeks after the initiation of exposure to Adriamycin® and X‐irradiation. The maximum percentages of YFF per 1,000 sperm for each individual in this exposed group were analyzed by Wilcoxon's distribution free rank sum test using a one‐sided alternative. The exposed individuals' maximum YFF percentages were statistically significantly increased when compared to the maximum YFF values of the non‐ exposed controls. The individuals exposed to the nematocide DBCP also exhibited a statistically significant increase in the number of sperm con‐ taining two Y chromosomes as determined by chi‐square analysis with one degree of freedom (P < 0.01).

Data presented herein show statistically significant increases in the incidence of double Y chromosomes as measured by the presence of YFF sperm following exposure to Adriamycin®, X‐irradiation, and DBCP. It is suggested that men who have a history of antineoplastic therapy could be evaluated for evidence of Y‐Y nondisjunction with this method. In the event of an increased YFF sperm level, genetic counseling and amniocentesis should be made available to the spouse where pregnancy has occurred. Further, be‐ cause this procedure measures gametic mutation, is relatively simple, and is noninvasive, it should be considered for inclusion as part of a battery of medical tests for monitoring industrial populations.
Test of carcinogenicity of quercetin, a widely distributed mutagen in food
Authors: Saito Daizo , Shirai Atsuko , Matsushima Taijiro , Sugimura Takashi , Hirono Iwao
Published: February 2006

The carcinogenicity of quercetin, a flavonol, was tested in six‐week‐old ddY mice of both sexes. Groups of 38 males and 35 females were given pellet diet containing 2% quercetin throughout their life span. As controls, 16 males and 15 females were given basal diet. Animals in both test and control groups developed leukemia and tumors of the lung, forestomach, mammary gland, adrenal, and soft part tissues. In addition, some animals in groups treated with quercetin developed tumors of the heart, liver, salivary gland, ovary, and uterus. The incidences of these tumors in test and control groups were not statistically different.
Teratogenicity and embryotoxicity of nickel carbonyl in Syrian hamsters
Authors: Sunderma F. William , Shen Samuel K. , Reid Marilyn C. , Allpass Patricia R.
Published: February 2006

Nickel carbonyl was administered to groups of pregnant hamsters by inhalation (0.06 mg Ni(CO)4/liter/15 min) on days 4, 5, 6, 7, or 8 of gestation. The dams were killed on day 15 of gestation, and the fetuses were examined for malformations. Exposure to Ni(CO)4 on days 4 or 5 of gestation resulted in malformations in 5.5% (8/146) and 5.8% (10/171) of the progeny, respectively (P < 0.05, versus 0/95 in controls). The proportions of litters with malformed fetuses were 33% (4/12) and 24% (4/17) in dams exposed to Ni(CO)4 on days 4 and 5 of gestation (P < 0.05, versus 0/9 in litters of control dams). Progeny of dams exposed to Ni(CO)4 on days 4 and 5 included 9 fetuses with cystic lungs, 7 fetuses with exencephaly, 1 fetus with exencephaly plus fused rib, and 1 fetus with anophthalmia plus cleft palate. Hemorrhages into serous cavities were found in 18% (26/146) and 25% (42/171) of fetuses of dams exposed to Ni(CO)4 on days 4 or 5 of gestation. Such hemorrhages were not observed in controls. In progeny of dams exposed to Ni(CO)4 on days 6 or 7 of gestation, there was 1 fetus with fused ribs and there were 2 fetuses with hydronephrosis. In another experiment, pregnant hamsters were exposed to inhalation of Ni(CO)4 (0.06 mg/liter/15 min) on day 5 of gestation; these dams were permitted to deliver their litters and to nurse their pups. On the day of delivery, there was no significant difference in the average number of live pups in the Ni(CO)4‐exposed litters compared to control litters. Neonatal mortality was increased in Ni(CO)4‐exposed litters; by day 4 postpartum, the number of live pups averaged 7.6 ± 1.5 in Ni(CO)4‐exposed litters (P < 0.01 versus 9.6 ± 1.8 pups in control litters). This study demonstrates that Ni(CO)4 is teratogenic and embryotoxic in Syrian hamsters.
Detection of urinary metabolites of 2‐acetylaminofuorene by cultured hamster embryo cells and high performance liquid chromatography
Authors: Lebherz William B. , Shaikh Badaruddin , Pienta Roman J.
Published: February 2006

Urine was collected from Sprague‐Dawley rats injected intraperitoneally with a suspension of 2‐acetylaminofluorene (AAF) or with an inert suspension vehicle. Morphological transformation was observed in clonal cultures of secondary passage hamster embryo cells (HEC) after the addition of AAF‐treated rat urine or concentrated urine extracts prepared by incubating the urine with β‐glucuronidase. No morphological alteration was observed in cultures treated with urine (or urine extracts) from rats injected with the suspension vehicle alone. The presence of N‐hydroxy‐ and ring‐hydroxylated metabolites of AAF in the AAF‐treated concentrated urine extract was confirmed by highperformance liquid chromatography (HPLC).
Mutagenicity of vinyl compounds in salmonella typhimurium
Authors: Lijinsky W. , Andrews A. W.
Published: February 2006

Eighteen compounds structurally related to the mutagenic carcinogen vinyl chloride have been tested for mutagenicity in five strains of Salmonella typhimurium. Acrolein, acrolein diethylacetal, acrylonitrile, allyl alcohol, allyl bromide, crotonaldehyde, crotyl alcohol, and vinyl bromide were mutagenic with one or more strains; acrolein bisulfite, acrolein oxime, acrylamide, acrylic acid, allylamine, trans‐cinnamaldehyde, trans‐cinnamic acid, crotonic acid, methyl methacrylate, and vinyl acetate were not mutagenic. The mutagenicity tests were performed both without and with activation by rat and hamster liver microsomal preparations (S9). All compounds were tested in the plate incorporation and/or liquid preincubation tests except vinyl bromide, which was tested as a gas.
Kinetics of uptake and biliary excretion of benzo(α)pyrene and mutagenic metabolites in isolated perfused rat liver
Authors: Forti G. Cantelli , Trieff N. M.
Published: February 2006

An isolated liver perfusion system was used as a simplifying tool to study the metabolism and excretion of benzo(α)pyrene (BP) as a prototype carcinogen/mutagen. Phenobarbital (PB) was used to induce liver microsomal enzymes in Sprague‐Dawley male rats prior to isolated liver perfusion. Control livers were run simultaneously using generally tritiated (G‐3 H)BP/BP as substrate in the perfusion medium. Both biliary excretion and liver weight were increased in the induced compared to control liver, but biliary flow when corrected for liver weight is statistically the same for both control and PB‐induced livers. The excretion rate of radioactivity in the bile is always higher for PB‐induced than for control liver (maximum radioactive excretion at 1 hr). There is a more rapid radioactivity removal in the liver perfusion medium for PB‐induced than for control livers. Data are explained by increased metabolism of BP in induced liver leading to the presence of more polar metabolites undergoing preferential biliary excretion than in the control liver. Results support in vivo experimental data. Extracts from liver and bile were tested for microbial mutagenicity by the Ames test (TA 100) after TLC separation. The control liver shows virtually no mutagenicity in bile, only in TLC fractions from the liver. The PB‐induced liver shows significant mutagenicity in several TLC fractions in both bile and liver. The net effect of induction is to produce more mutagenic metabolites of BP, excreted in the bile, and presenting a significant exposure of carcinogens/mutagens, and consequent hazard to man.
The mutagenicity of 45 nitrosamines in Salmonella typhimurium
Authors: Andrews A. W. , Lijinsky W.
Published: February 2006

The correlation between mutagenicity in the rat liver microsome–mediated Salmonella Mutagenicity Assay of Ames and carcinogenicity in rats was examined with three groups of nitrosamines. Qualitatively the correlation was good, but there was poor correlation between mutagenic potency and carcinogenic potency. Of 23 cyclic nitrosamines, 19 were carcinogenic and mutagenic, and two were carcinogenic but not mutagenic, and the carcinogenicity studies of the remaining two are not complete. Of six symmetrical aliphatic nitrosamines, five were carcinogenic and mutagenic while only one carcinogen was not mutagenic. The greatest discrepancy occurred among 16 asymmetric nitrosamines, where 11 were both carcinogenic and mutagenic, four were carcinogenic but nonmutagenic, and one carcinogenicity study is incomplete.
The Heritable Translocation Assay: Its relationship to assessment of genetic risk for future generations
Authors: Bishop Jack B. , Kodell Ralph L.
Published: February 2006

The Heritable Translocation Assay (HTA) is an in vivo mammalian mutation test which measures specific clastogenic damage. It can be subjected to rigorous statistical scrutiny and detects a type of heritable genetic damage that is known to contribute to the human disease burden. Because of these factors the HTA is one of the major tests of the battery used in regulatory evaluation of potential environmental mutagens. Although tests for detecting inherited translocations have been developed and improved over the past 40 years, further emphasis on several statistical considerations affecting the precision and reliability of HTA animal model data applied to regulatory hazard evaluation and risk assessment seems warranted. In this paper we have presented a brief history and the basic methodology for the HTA. Statistical concepts related to the design and implementation of HTA studies, such as the incorporation of false classification error probabilities in determination of required sample size and use of the single sample binomial versus Fisher's exact test for analysis of data, have been examined in detail. We have also suggested a simplified method of extrapolating HTA animal data to man similar to that used with carcinogenesis data and propose other developmental needs and perspectives for application of this assay to regulatory assessment of environmental mutagens.
Aggregation patterns of fetal rat brain cells following exposure to X‐irradiation
Authors: Shoji R. , Suzuki K. , Lee I. P.
Published: February 2006

In our search for a simplified in vitro test system to assess the teratogenic effects of physical factors, we studied the effects of total maternal body X‐irradiation on aggregation patterns of enzymatically isolated fetal rat brain cells and on ultrastructural aggregate changes. The fetal brain cells were derived from day 14 gestation fetuses of pregnant Sprague‐Dawley (CD strain) rats exposed to X‐irradiation (25˙200 R) one hour prior to sacrifice. Notable changes in the cell aggregates following X‐irradiation included a reduction in cell aggregate size and an increase in number. The frequency of cell aggregates was higher in the treated than in the control group, and the mean diameter of cell aggregates was inversely related to increasing X‐irradiation doses. Transmission electron microscopy revealed in isolated cells features of degenerative processes which were similar to those found in intact fetal brain lesions caused by maternal X‐irradiation. Furthermore, scanning electron microscopy revealed that inhibition of cell aggregation following X‐irradiation could probably be attributed to inhibition of membrane filopodia development and a consequent failure of cell aggregates to fuse into a greater cell aggregate mass. These results suggest that the membrane factors which influence cell aggregation may be a useful parameter to assess early effects of X‐irradiation‐induced brain deformity. Presently, the cell aggregation culture system is being further evaluated as a short term test system for environmental teratogens.
Teratogenicity of valproic acid: In vivo and in vitro investigations
Authors: Kao J. , Brown N. A. , Schmid B. , Goulding E. H. , Fabro S.
Published: February 2006

The in vivo teratogenic potential of valproic acid was evaluated in the CD‐1 mouse. Dose selection was based on the estimated lethality curve and several doses were evaluated. Treatment during early organogenesis (Days 8–10) produced a dose related increase in congenital abnormalities which was accompanied by a decrease in fetal weight, Rib and vertebral abnormalities together with exencephaly were the most common defects. Greater embryo mortality was apparent following teatment during late organogenesis (Days 11–13), but the surviving fetuses were less sensitive to the dysmorphogenic effects and cleft palate was now the most common defect, Early neurula stage mouse (Day 9) and rat (Day 10) embryos were cultured in the presence of valproic acid (0.6–1.8 mM) for 48 hr. There was significant reduction in growth which was accompanied by a variety of dysmorphogenic effects. Irregular segmentation of somites together with incomplete and irregular fusion of brain folds were most evident. These were observed at concentrations of valproic acid where no adverse effects on the yolk sac circulatory system were apparent. These in vitro effects appear to be comparable to the in vivo observations and suggest a direct teratogenic action of valproic acid on the developing rodent embryo.
The lack of estrogen control of rodent alphafetoprotein levels
Authors: Sheehan Daniel M. , Branham William S.
Published: February 2006

Alphafetoprotein (AFP) binding of estrogens is thought to protect fetal and neonatal rats from the toxic effects of high maternal estrogen levels. Since the plasma concentrations of several other steroid binding proteins are ligand regulated, we tested the hypothesis that estrogens regulate plasma AFP levels. A batch Sephadex equilibrium binding assay and Scatchard analysis were used to quantitate AFP in plasma of both adult mice (C3H/HeN MTV−) chronically fed diethylstilbestrol (DES) or estradiol (E2) and neonatal rats (CD) injected daily with E2 for the first 15 days of life. Estrogen treatment of adults did not increase AFP above the nondetectable adult levels, nor did it alter the plasma disappearance curve of neonatal AFP. In both adults and neonates, other estrogen responses were noted, demonstrating that the failure of biologically active estrogens to alter AFP levels is not due to insufficient amounts of estrogen. We suggest that estrogens do not alter patterns of AFP synthesis and degradation in the rat, a finding which contrasts with the general observation that steroids regulate the levels of their binding proteins.
Mutation induction and killing of prokaryotic and eukaryotic cells by 8‐methoxypsoralen, 4,5′‐dimethylangelicin, 5‐methylangelicin, 4′hydroxymethyl‐4,5′‐dimethylangelicin
Authors: Pani B. , Babudri N. , Venturini S. , Tamaro M. , Bordin F. , Monti‐Bragadin C.
Published: February 2006

Cell killing and induction of mutations were studied in V79 Chinese hamster cells and in Escherichia coli WP2 and WP2 uvrA as a result of treatment with four furocoumarins and near ultraviolet light (NUV). The results obtained on V79 Chinese hamster cells showed that the three angelicin derivatives tested have mutagenic as well as cytotoxic activity, although at different levels; 8‐methoxy‐psoralen (8‐MOP), which is the most cytotoxic drug, induces the lowest number of 6‐thioguanine (6‐TG) resistant mutants as a function of surviving fraction. The three angular furocoumarins, 4,5′‐dimethylangelicin, 5‐methylangelicin, and 4′‐hydroxymethyl‐4,5′‐dimethylangelicin, were found not to be bactericidal and only slightly mutagenic in excision‐proficient Escherichia coli. They were much more active in inducing cell death and mutations in Escherichia coli uvrA. On the contrary 8‐methoxypsoralen displayed mutagenic and cytotoxic activity in E. coli WP2 uvr+ as well as in E. coli uvrA. This study confirms that (1) mono‐adducts are efficiently repaired in excision‐proficient bacterial strains and (2) the nature of substituents could affect biological properties, such as mutagenic and cytotoxic activity, of furocoumarins.
Plasma membrane alterations due to promoters and antipromoters
Authors: Braun Andrew G. , Buckner Christine A. , Nichinson Bradley B.
Published: February 2006

The attachment of tumor cells to plastic surfaces coated with concanavalin A is inhibited by agents that either increase or decrease the apparent microviscosity of the plasma membrane. Since some promoters have been reported to decrease the plasma membrane microviscosity of treated cells, we have tested whether these agents inhibit tumor cell attachment. All 13 promoters tested were found to inhibit attachment at nontoxic concentrations. The apparent microviscosity of treated plasma membrane was reduced in the 11 cases where a measurement was possible. Eight of nine nonpromoting agents did not inhibit attachment. The exception, Aroclor 1254, was inhibitory on prolonged incubation. Finally, 10 of 13 antipromoting agents were found to also inhibit attachment. The apparent membrane microviscosity was increased following treatment with these inhibitory antipromoters. Thus, a consistent feature of promoter and antipromoter treatment is an alteration of the functional and the apparent fluid properties of the plasma membrane. These characteristics may be useful in the design of an in vitro assay for promoting activities in environmental samples.
Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing
Authors: Stuckhardt J. L. , Poppe S. M.
Published: February 2006

A method for examining the viscera of rat and rabbit fetuses is described. Techniques used in detecting visceral alterations in rats and rabbits for routine teratogenicity screens have varied over the years. The method used should be quick and simple but at the same time must be accurate, reliable, and comprehensive. The procedure used in this lab is a complete and systematic necropsy of the fresh fetus requiring minimum equipment and time. The examination can be done immediately following cesarean section and yields an intact skeleton which can subsequently be processed for skeletal examination. The fresh specimen and the natural coloration of in situ organs makes color photography of visceral alterations clear and concise. Any lesion can be appropriately fixed for histopathic examination. This technique begins with the examination of the organs in the abdominal cavity and proceeds to the thorax. Of special interest is the procedure used to inspect the internal anatomy of the fresh fetal heart. A description of the internal examination of both rat and rabbit heads and eyes is also included.
Mutagenic and cytogenetic analyses of organic extracts of simulated runoffs from model coal piles
Authors: Stahl Ralph G. , Arrighi Frances E. , Matney Thomas S. , Bernard Gary S. , Johnston Dennis A.
Published: February 2006

The use of coal as a fuel in utility and other industries in the United States is increasing. Typically, these utilities store their coal outdoors in large piles, and rainfall on the piles produces a runoff containing hazardous inorganic and organic materials. Four coals of varying sulfur contents, all used for fuel in the United States, were tested. Organic materials were extracted from simulated runoffs of model coal piles and were tested for mutagenicity with a Salmonella/microsomal assay and for clastogenicity and cytotoxicity in Chinese hamster ovary cells. The extracts of the high‐sulfur coals and the lignite were more mutagenic and clastogenic than extracts from the low‐sulfur coal.
Two‐phase teratology study with the synthetic prostaglandin ONO‐802 given intravaginally to rats
Authors: Petrere Judith A. , Humphrey Ronald R. , Sakowski Raymond , Fitzgerald James E. , de la Iglesia Felix A.
Published: February 2006

The synthetic prostaglandin ONO‐802 was administered intravaginally to Sprague Dawley rats at doses of 1.0, 0.5, and 0.125 mg/kg on days 6 through 15 of gestation. A vehicle control group was treated with pessaries that did not contain the drug while another group remained untreated. Body weight, food, water consumption, and clinical signs were monitored during the experiment. In Phase One, 20 pregnant animals from each group were sacrificed at term, major organs were weighed, and litter and fetal data were collected. In Phase Two ten dams per group were allowed to deliver their litters, and the offspring were evaluated for survival, growth, developmental signs, and physiological function. Selected F1 offspring were retained to assess learning and emotional behavior or reproductive capacity.

Administration of either 0.5 or 1.0 mg/kg of ONO‐802 resulted in a slight reduction in food consumption and body weight gain. Water consumption was increased both during and after the dosing period for the mid and high dose dams. Significantly increased weights for the heart, lungs, liver, adrenals, and ovaries and decreased weights for the thymus gland were noted at term sacrifice of the 1.0 mg/kg dams, whereas the 0.5 mg/kg group had increased weights of the adrenals and ovaries only. Litter parameters were unaffected by treatment. Weights of the female fetuses of the 1.0 and 0.5 mg/kg groups were significantly reduced when compared to controls. There were no significant drug‐related abnormalities among the F1 offspring and no evidence that treatment of the F0 dams affected the development, behavior, or reproductive performance of the F1 offspring. Thus, ONO‐802 was not teratogenic when given to rats by the intravaginal route.
Mutagenic, cytotoxic, and teratogenic effects of 2‐acetylaminofluorene and reactive metabolites in vitro
Authors: Faustman‐Watts Elaine M. , Yang Hsueh Ying L. , Namkung Moses J. , Greenaway Jean C. , Fantel Alan G. , Juchau Mont R.
Published: February 2006

The embryotoxic, mutagenic, and cytotoxic properties of 2‐acetylaminofluorene (AAF) and two of its reactive metabolites, N‐acetoxy‐2‐acetylaminofluorene (AAAF) and 2‐nitrosofluorene (NF) were assessed in vitro. A combined embryo culture/biotransformation system was used to determine the ability of these compounds to produce embryonic malformations, growth retardation, and/or embryolethality. Salmonella typhimurium auxotrophs (his−) were utilized to measure the mutagenic and cytotoxic potentials of these compounds.

The parent compound, AAF, did not produce embryonic malformations or mutagenicity in the absence of an added cytochrome P‐450‐dependent monooxygenase system. Both metabolites produced each of the measured toxic effects without supplementation of a bioactivation system. However, the three chemicals each elicited a different spectrum of malformations. Bioactivated AAF produced neural tube abnormalities, whereas embryos treated with AAAF primarily exhibited prosencephalic malformations, and NF produced abnormalities of axial rotation or flexure. NF was approximately ten times more potent than AAAF as a direct‐acting mutagen but only slightly more active in producing embryonic malformations in vitro. The results indicated that differential effects on the various measured parameters could be produced by these chemicals. The results indicated further that neither NF nor AAAF appeared to be individually responsible for the neural tube abnormalities generated by biotransformed AAF.
Mouse limb bud development in submerged culture: Quantitative assessment of the effects of in vivo exposure to retinoic acid
Authors: Kwasigroch T. E. , Skalko R. G. , Church J. K.
Published: February 2006

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose‐dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid‐induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose‐dependent decrease in area of both fore‐ and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.
Developmental toxicity during the preimplantation period: Embryotoxicity and clastogenic effects of chlorambucil in the rat
Authors: Giavini E. , Bonanomi L. , Ornaghi F.
Published: February 2006

To evaluate the toxicological effects of chemicals on preimplantation mammalian embryos, pregnant rats were treated with chlorambucil (0, 3, 6, and 12 mg/kg IP) on day 1, 2, or 3 of gestation (positive vaginal smear = day 0). Blastocysts were collected on day 4 and evaluated for gross morphology, cell number, and mitotic index. Some females treated on day 3 post coitum were sacrificed on day 20 and fetuses were evaluated for teratogenic effects. On day 4 of gestation a dose‐related reduction of cell number/embryo was recorded; this effect was most manifest with treatment on day 3 post coitum. The mitotic index was significantly higher in all treated groups. A dose‐related increase in “micronucleus‐like bodies” has been observed in treated embryos. A comparison with the micronucleus test in the bone marrow revealed a major sensitivity of the embryonic material. No malformations have been observed in fetuses at term, but their weights were significantly reduced and a dose‐related increase of postimplantation loss was recorded in treated females.
Development of a mouse embryo limb bud cell culture system for the estimation of chemical teratogenic potential
Authors: Guntakatta M. , Matthews E. J. , Rundell J. O.
Published: February 2006

High‐density cultures of mouse embryo limb bud cells differentiate and synthesize an extracellular matrix containing sulfated proteoglycans. Since these in vitro events are related to those that occur during fetal development, we have investigated the use of cultured limb bud cells for the analysis of the activities of chemical teratogens. We have established the conditions for the use of the radiochemicals 35SO and 3H‐thymidine for the assessment of chemical effects on sulfated proteoglycan and DNA synthesis, respectively, in mouse limb bud cells. By performing double‐labeling experiments in the presence of test chemicals, the preferential inhibition of either proteoglycan synthesis or DNA synthesis could be demonstrated for 19 of 22 known mouse teratogens tested. The five nonteratogens tested did not cause significant differences in the inhibition of either macromolecular class. The overall predictive accuracy of the system was ∼89%, and the false‐negative rate was ∼14.8%. No false positives were observed. These data showed that this cell culture system differentiated between the activites of a limited set of selected mouse teratogens and nonteratogens, suggesting that cultured mouse embryo limb bud cells may constitute the basis for the development of a powerful tool for analyses of chemical teratogenic potential.
Short‐term bioassays of fractionated emission samples from wood combustion
Authors: Alfheim I. , Becher G. , Hongslo J. K. , Lazaridis G. , Löfroth G. , Ramdahl T. , Rivedal E. , Salomaa S. , Sanner T. , Sorsa M.
Published: February 2006

Extracts of an emission sample from wood burning, consisting of particles and volatiles, have been fractionated on an HPLC silica gel column into five fractions of increasing polarity. Nonfractionated samples and the individual fractions have been tested in three different short‐term bioassays: the Ames Salmonella assay, the sister chromatid exchange (SCE) induction‐test in Chinese hamster ovary cells (CHO), and the cell transformation test on Syrian hamster embryo (SHE) cells.

Most of the total activity was found in the volatile part of the sample with all three bioassays, whereas the particle extract had the highest activity per unit mass extracted. The second most polar fraction contained most of the mass and was also highly active in all assays. The most polar fraction was very potent in the Salmonella assay, but showed only a weak response in the eukaryotic bioassays. Storage of the samples for several months at 0°C revealed that the bacterial mutagens present in the most polar fraction were labile; the mutagenicity was almost totally lost after 1 year's storage.
The effects of phenylalanine on cultured rat embryos
Authors: Walsh D. A. , Christian Z. H.
Published: February 2006

The offspring of mothers with untreated classic phenylketonuria (PKU) have shown a high frequency of microcephaly, mental retardation, pre‐ and postnatal growth retardation, and birth defects. The aim of this study was to determine whether phenylalanine (phe) is the teratogenic agent in maternal PKU.

To observe the direct effects of phe on organogenesis, embryos of 9.5‐day pregnant rats were cultured for 48 h in the presence of phe at concentrations of 0.1 to 6.0 mM. Within this range no morphological abnormalities occurred in exposed embryos, when compared to control embryos. However there was a reduction (P ≤ 0.05) in embryonic protein content and somite number at the highest concentration of phe (6.0 mM). This does not preclude the longer‐term effects of phe at later stages of gestation.

To examine phe transport into the embryo in response to elevated serum phe levels, 3H‐phe uptake studies were undertaken. These showed that 3H‐phe from the culture serum is incorporated rapidly into the free amino acid pools and embryonic protein. At serum concentrations of 1.4 mM or higher, phe saturation occurs in the cellular pools of the embryo. Amino acid analysis of the exocoelomic fluid showed that when embryos were cultured for 48 h in serum containing 3.45 mM phe, the total amino acid concentration was maintained near the control levels (16 mM). Of this, 27% (4.26 mM) was contributed by phe, and all other amino acids, except methionine, were decreased with respect to control levels.
Induction of chromosome aberrations and specific locus mutation but not sister chromatid exchanges in Chinese hamster ovary cells by neocarzinostatin
Authors: Au W. W. , O'Neill J. P. , Wang W. , Luippold H. E. , Preston R. J.
Published: February 2006

The induction of chromosome aberrations, sister chromatid exchanges (SCE), cytotoxicity, and 6‐thioguanine‐resistant mutation by neocarzinostatin (NCS) in Chinese hamster ovary cells was analyzed. It was observed that within the same concentration range of 0.01–0.1 μ/ml NCS, the drug induced a significant increase in all analyzed end‐points except in SCE frequencies. There was no increase in SCE frequencies even when the cells were treated at the G1/S border in the first cell cycle and when aberrations were observed in the same cell showing a second cycle differential staining pattern. Our study indicates that the cellular damage induced by NCS leads to expression in chromosome aberrations, cytotoxicity, and mutagenicity but not in sister chromatid exchanges.
DNA damage and repair in mouse embryos following treatment transplacentally with methylnitrosourea and methylmethanesulfonate
Authors: Jirakulsomchok Saowalak , Yielding K. Lermone
Published: February 2006

Mouse embryos were labeled in vivo at 10 1/2–12 1/2 days of gestation with [3H]‐thymidine and subjected to DNA damage using x‐ray, methylmethanesulfonate, or methylnitrosourea. DNA damage and its repair were assessed in specific cell preparations from embryos isolated at intervals thereafter using the highly sensitive method of nucleoid sedimentation, which evaluates the supercoiled state of the DNA. Repair of x‐ray damage was demonstrated using trypsin‐dispersed cells from whole embryos and from homogenized embryonic liver to show the validity of the analytical approach. The effects of the highly teratogenic methylnitrosourea and the much less teratogenic methylmethanesulfonate were compared in the targeted limb buds using equitoxic doses of the two alkylating agents. DNA supercoiling was fully restored after 24 hr in limb bud cells damaged with methylmethanesulfonate, while as much as 48 hr were required for full repair of methylnitrosourea damage. These results demonstrated the feasibility of studying DNA repair in embryonic tissues after damage in vivo and suggest that the potency of methylnitrosourea as a teratogen may be correlated with a prolonged period required for complete repair of DNA.
Assay of mutagens in aqueous fecal extracts with a modified ames Salmonella test
Authors: Shaw Roger , Andrews A. W. , Riggs Charles W.
Published: February 2006

Mutagenicity with Salmonella strain TA 100 can be determined by an island test in which only an isolated portion of a pour plate is spotted with a sample. The magnitude of the ratio of the numbers of revertant colonies on the treated and untreated parts of the plate reflects the potency of the mutagen. Five of six chemicals tested yielded statistically significant and generally linear dose‐response curves. Minimum detectable mutagenic doses for four compounds calculated from the dose‐response curves showed that the island test generally required less material for detection of mutagenicity than the liquid pre‐incubation procedure. The island method was primarily designed to test the mutagenicity of aqueous extracts of human stool samples. Dose‐responses were obtained for six such samples, and in two samples the amount of material needed for a positive response was significantly less than that required in the liquid pre‐incubation method.
Effects of vitamins A, C, and E on aflatoxin B1‐induced mutagenesis in Salmonella typhimurium TA‐98 and TA‐100
Authors: Raina Vijay , Gurtoo H. L.
Published: February 2006

The effects of retinoids (vitamin A analogs) and vitamins C and E on the aflatoxin B1 (AFB1)‐induced mutagenesis in Salmonella typhimurium TA‐98 and TA‐100 were investigated. The bioassay was performed under conditions that permitted the effects of vitamins on carcinogen metabolism to be assessed separately from effects on the expression of the mutated bacterial cell.

Both retinoic acid and retinol inhibited (up to 50%) AFB1‐induced mutagenesis in S. typhimurium TA‐98, but only retinol inhibited (up to 75%) mutagenesis in TA‐100. Retinoic acid inhibition of mutagenesis in S. typhimurium TA‐98 was pronounced over a wide concentration range (i.e., 2 × 10−10 to 2 × 10−8 M) however, at the higher concentrations (i.e., 2 × 10−8 to 2 × 10−6 M range) the predominant effect was the inhibition of the metabolism of AFB1 to its mutagenic metabolites. Vitamin E was more potent in inhibiting the expression of AFB1‐induced mutagenesis than vitamin C. However, the major inhibitory effects of vitamin E were related to the metabolism of AFB1, whereas vitamin C was inhibitory at both metabolic and the post‐metabolic levels of the AFB1 mutagenesis assay.

The results of these investigations suggest that vitamins A, C, or E inhibit both AFB1 metabolism to its mutagenic metabolites as well as the expression of AFB1‐induced mutated bacterial cells.
Vascular anomalies and pyrimethamine‐induced malformations in the rat
Authors: Tangapregassom Anne‐Marie , Tangapregassom Marie‐Joseph , Horvath Cécilia , Trecul Michèle , Boucher‐Ehrensperger Maud , Petter Claude
Published: February 2006

Pyrimethamine injected into the rat on days 12, 13, and 14 of gestation induces several types of malformations, including limb and facial deformities. These malformations appear in the same location as the hemorrhages and hematomas induced by this substance. In order to visualize the extent of the vascular disorders in the affected areas, a polymerizable substance was injected intravenously into the fetus. After pyrimethamine treatment, the fetal vascular network was simplified and reorganized in a different way according to the severity of the malformations. The appearance of vascular pathology, followed by malformations in the same regions, raises the hypothesis of a vascular cause of fetal defects.
Supraadditive formation of micronuclei in preimplantation mouse embryos in vitro after combined treatment with X‐rays and caffeine
Authors: Müller W.‐U. , Streffer C. , Wurm R.
Published: February 2006

The influence of caffeine (0.1 or 2 mM), X‐rays (0.24 Gy [ = 25 R] or 0.94 Gy [ = 100 R]), or of a combination of both on the formation of micronuclei in early stages of preimplantation mouse embryos in vitro was studied. X‐rays as well as caffeine induced micronuclei. The dose‐effect curve after irradiation was linear for the dose range measured (0–3.76 Gy; = 0–400 R). Caffeine did not induce micronuclei if the concentration was 1 mM or less; between 1 mM and 7 mM, however, there was a linear increase in the number of micronuclei. A considerable enhancement of the number of radiation‐induced micronuclei was observed when irradiation of the embryos was followed by a treatment with caffeine. Not only was the sum of the single effects exceeded by the combination effects, but the combination results even lay in the range of supraadditivity of the envelope of additivity.
Analysis of the activity of DNA, RNA, and protein synthesis inhibitors on Xenopus embryo development
Authors: Courchesne Cheryl L. , Bantle John A.
Published: February 2006

The teratogenic and growth‐inhibiting potential of DNA, RNA, and protein synthesis inhibitors was explored using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Endpoints measured in 96‐h static tests were survival, malformation, ability to swim, skin pigmentation, stage of development, and growth. The DNA synthesis inhibitors hydroxyurea, cytosine arabinoside, and ethidium bromide proved to be teratogenic by the severity of malformations induced. Hydroxyurea gave an LC50 of 1.82 mg/ml, an EC50 (malformation) of 0.43 mg/ml, while the values for cytosine arabinsode were 5.41 and 0.76, respectively. The values for ethidium bromide were 0.05 and 0.035. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide were more embryolethal than teratogenic but significantly inhibited growth as determined by head‐tail length measurements. Actinomycin D caused severe malformations, while cycloheximide caused relatively minor abnormalities. The LC50 for actinomycin D was 1.89 mg/ml, while the EC50 (malformation) was 2.17 mg/ml. For cycloheximide, the values were 1.59 and 1.19, respectively. FETAX advantages include rapid data collection, the ability to measure stage‐dependent effects, and the ability to use a large number of embryos to obtain excellent doseresponse curves with narrow confidence limits. Disadvantages include lack of a metabolic activation system, absence of a placental relationship, and the inability to detect specific abnormalities such as limb defects in 96 h.
Propyldazine is mutagenic in Salmonella typhimurium and Escherichia coli: Distinct specificity for strains TA1537 AND TA97
Authors: Glatt Hansruedi , Oesch Franz
Published: February 2006

The antihypertensive drug propyldazine (Atensil) was demonstrated to be muta‐ genic with auxotrophic mutants of Salmonella typhimurium and Escherichia coli. Addition of liver S9 mix (postmitochondrial supernatant fraction supplemented with an NADPH‐generating system) had little, if any, effect on the mutagenicity. The mutagenicity showed an unusual pattern of strain specificity. Increased fre‐ quencies of reversion were observed with all strains whose auxotrophy was caused by frame‐shift mutations: the number of revertant colonies per plate from S. typhimurium TA98, TA1538, TA97, and TA1537 was increased up to 5‐, 9‐, 43‐, and 160‐fold, respectively, above background. Among the strains that became auxotrophic by substitution mutations, S typhimurium TA102, E. coli WP2, and E coli WP2 uvrA yielded positive results (twofold above background). S. typhimurium TA1535 and TA100 were not reverted by propyldazine.

It should be noted that propyldazine, due to its low toxicity and good solubility, could be tested up to very high doses. Hence, although quite impressive mutagenic effects occurred, the mutagenic potency was moderate even in the most responsive strains, TA1537 and TA97 (about 0.3 and 1.0 revertants per nmole, respectively). With the limitation that the strain specificities were different, the mutagenic potency of propyldazine was in the same order of magnitude as that of hydralazine and dihydralazine, two related antihypertensive drugs which were already known to be mutagenic. In our hands, both compounds were mutagenic in S typhimurium TA1535, TA100, TA1537, and TA98. These results differ from data in the literature in that we found clear but weak effects even with strains for which others have reported negative results.
A critical comparison of the freehand razor‐blade dissection method according to Wilson with an in situ sectioning method for rat fetuses
Authors: Sterz H. , Lehmann H.
Published: February 2006

Two methods of dissecting rat fetuses are compared: the generally well‐known freehand razor‐blade dissection method according to Wilson and an in situ sectioning method (ISM). For this purpose the substance EGYT 1978 was used, which induces—inter alia—cardiovascular anomalies. Thirty‐nine gravid rats were given either the test drug (EGYT 1978, 400 mg/kg body weight) or the vehicle orally from the 6th to the 15th day of gestation. The fetuses were assigned randomly to either dissection group. Both methods of examination revealed approximately the same number of anomalies per organ system. A comparison of the types of anomaly found showed, however, relevant differences in the nature of the changes revealed by the two methods. The in situ sectioning method was clearly superior to the freehand razor‐blade dissection method for the detection of discrete anomalies of the cardiovascular system. We therefore recommend the in situ sectioning method as the method of choice for examining small fetuses from teratological experiments.
Teratogenic profile of retinylidene methyl nitrone and retinol in Swiss‐Webster mice
Authors: Willhite Calvin C. , Balogh‐Nair Valeria
Published: February 2006

A single oral dose of 75 mg/kg of all‐trans‐retinol or all‐trans‐retinylidene methyl nitrone, retinoids with potential cancer chemopreventive properties, on one of five successive days of embryogenesis resulted in a shift in the pattern of developmental anomalies in fetal mice. Treatment on days 7, 8, or 9 with retinol primarily induced malformations of the head whereas treatment on day 11 induced bilateral forelimb reduction defects. Treatment on day 8 with either retinoid produced the highest in utero death rate. Intubation of either retinoid on day 10 failed to induce a significant increase in the number of litters containing offspring with malformations, and the embryonic death rate declined to control values. The malformations induced by administration of either retinoid were similar, but retinol was always associated with a higher total percentage of malformed offspring. The similar teratogenic profile of these two retinoids may be related to their in vivo biotransformation to all‐trans‐retinoic acid.
An analysis of lung cancer risk from exposure to hexavalent chromium
Authors: Braver Elisa R. , Infante Peter , Chu Kenneth
Published: February 2006

Lung cancer risk in relation to airborne levels of hexavalent chromium was analyzed for chromium chemical production workers studied by Hayes, Lilienfeld, and Snell. Hayes et al [18] had observed statistically significant increases in lung cancer mortality among chromium chemical production workers hired during 1945–1959 and followed to mid‐1977. A dose‐response was observed by Hayes et al. in that long‐term workers had a higher lung cancer risk than short‐term workers. Concurrent exposure data for the study plant were abstracted from the records of a local health department. The usual air concentration of hexavalent chromium was estimated as 413 micrograms per cubic meter (μg/m3) during 1945–1949. Cumulative exposure estimates for individual workers could not be developed with available information. Instead, cumulative exposures in terms of μg/m3‐years were estimated for groups of short‐term workers and long‐term workers (cumulative exposure = usual exposure level in μg/m3 × average length of exposure). For workers hired during 1945–1949, cumulative exposures were estimated as 670 and 3,647 μg/m3‐years for short‐term and long‐term workers, respectively. Like most estimates based on historical data, these exposure estimates are subject to uncertainty. Nevertheless, these results suggest a potential excess risk of death from lung cancer among U.S. workers exposed to the current permissible exposure limit (PEL) for hexavalent chromium of 52 μg/m3 because such workers could accumulate exposures (μg/m3‐years) similar to those associated with excess risk in Hayes et al's cohort. Moreover, many current workers are estimated to be exposed to levels above the PEL. Further exploration of the dose‐response relationship for chromium carcinogenesis is indicated.