bluuebunny/crossref_metadata_2025 huggingface dataset:
Karyotype, growth, and cell cycle analysis of human embryonic palatal mesenchymal cells: Relevance to the use of these cells in an in vitro teratogenicity screening assay
Authors: Welsch Frank , Stedman Donald B. , Willis William D. , Pratt Robert M.
Published: February 2006

Human embryonic palatal mesenchyme (HEPM) is an established cell line that is presently under investigation as an in vitro prescreening assay used to determine the teratogenic potential of chemicals. We describe here general growth characteristics, karyotype, and cell cycle analysis of these cells. HEPM cells had plating efficiencies of <95% and displayed notable contact growth inhibition following an exponential growth phase that lasted for approximately 6 days. These cells had the diploid karyotype of a female human embryo. The chromosomal complement showed no dramatic change between passage 5 and 14. Flow cytofluorometry analysis using bromodeoxyuridine (BrdU) pulse labeling and a direct immunofluorescence anti‐BrdU FITC probe revealed that the total cell cycle transit time was approximately 22 hr: the duration of G1 was 12.2 hr, S was 6.1 hr, and G2‐M lasted for 3.7 hr. The results indicate that HEPM cells met the criteria regarding karyotype stability that were assumed by the National Toxicology Program of the USA.
Failure of epoxide formation to influence carbamazepine‐induced teratogenesis in a mouse model
Authors: Finnell Richard H. , Mohl Virginia K. , Bennett Gregory D. , Taylor Stephen M.
Published: February 2006

The teratogenic potential of the anticonvulsant drug carbamazepine was determined following chronic oral administration in two inbred mouse strains (SWR/J and LM/Bc). The drug was administered in the animal's diet in concentrations equivalent to 0, 1,000, 1,500, or 2,000 mg/kg body weight, with treatment starting 2 wks prior to mating and continuing throughout gestation. Fetal examination failed to reveal a significant pattern of malformation in either strain at any treatment level. Levels of plasma carbamazepine and its metabolite, carbamazepine‐10, 11‐epoxide, were determined by high pressure liquid chromatography. There was no correlation with either of these compounds and the incidence of fetal abnormality. The inherent teratogenicity of carbamazepine is significantly lower than that of other anticonvulsant drugs that have been similarly tested in an animal model.
The induction of supernumerary ribs in rodents: Role of the maternal stress
Authors: Beyer Patricia E. , Chernoff Neil
Published: February 2006

Increased incidences of supernumerary ribs (SNR) are a relatively common finding in standard teratology bioassays, and previous studies have indicated a possible correlation between their occurrence and general maternal stress. The present study describes the effects of immobilization stress on the induction of supernumerary ribs. To isolate the sensitive period of SNR induction, Sprague‐Dawley rats and CD‐1 mice were treated with 300 and 1,500 mg/kg, respectively, of sodium salicylate on single days 7–11 of gestation. In the rat, day 10 was found to be the sensitive period of lumbar rib induction (32% SNR vs 10% on other days) while day 9 was critical in the mouse (71% SNR vs <29% on other days). In a second set of experiments, maternal stress was accomplished by restraining two groups of gravid females in the supine position for 12 hours on the predetermined sensitive day. One group was immobilized from 9 am to 9 pm, while the second group was restrained from 9 pm to 9 am. Concurrent controls were food and water deprived for similar periods. Additional untreated controls were also included. An increase in supernumerary ribs was noted in stressed mice but not in rats. The 9 am to 9 pm mouse group exhibited the highest increase in supernumerary ribs (41%) as well as significant incidences of fused ribs and exencephaly. A significant linear relationship between maternal weight loss during treatment and increases in supernumerary ribs was also noted. Results suggest that for some compounds, supernumerary ribs may be the indirect result of agent‐induced, generalized maternal stress in the CD‐1 mouse.
Glucocorticoid receptor‐mediated teratogenesis and cell proliferation in the limbs and face of the chick embryo
Authors: Pavlík Alfred , Novotná Božena , Jelínek Richard
Published: February 2006

The susceptibility of the face region of the chick embryo to the teratogenic action of intraamniotically injected hydrocortisone contrasts with the resistance of the limbs to its action while at the same time their dysmorphogenesis may be induced by other agents. Since glucocorticoid receptors were shown to mediate face teratogenesis, their development was investigated in freshly dissected limb buds of 3‐, 3.5, and 4‐day‐old chick embryos in comparison with the face region. The specific binding of 3H‐dexamethasone to molybdate‐stabilized glucocorticoid receptors was estimated by the dextran‐coated charcoal method and complemented by cytologic analysis of mitotic activity in control and hydrocortisone‐treated embryos. The glucocorticoid receptors were found in both organ anlagen already on day 3 when their concentration in femtomoles per microgram DNA was significantly higher in the face region. Accordingly, on day 3 intraamniotic hydrocortisone inhibited the mitotic activity in the face without affecting the developing limbs. On days 3.5 and 4 the concentration of glucocorticoid receptors was similar in both organ anlagen. Administration of hydrocortisone on day 4 induced mitotic depression in the face as well as in the limbs. However, the degree of inhibition apppeared to be dependent upon the actual mitotic rate. In the face region where the mitotic activity culminated at that time, the inhibition was much deeper and longer‐lasting than in the developing limbs characterized by continuous decrease of proliferation rate in controls. These findings are consistent with a view that glucocorticoid receptors are a prerequisite, but not the only factor in receptor‐mediated teratogenesis.
Evaluation of 60 chemicals in a preliminary developmental toxicity test
Authors: Hardin Bryan D. , Schuler Ronald L. , Burg JeAnne R. , Booth Gary M. , Hazelden Keith P. , MacKenzie Karen M. , Piccirillo Vincent J. , Smith Kirby N.
Published: February 2006

The number of chemicals in commerce which have not been evaluated for potential developmental toxicity is large. Because of the time and expense required by conventional developmental toxicity tests, an abbreviated assay is needed that will preliminarily evaluate otherwise untested chemicals to help prioritize them for conventional testing. A proposed short‐term in vivo assay has been used in a series of studies in which a total of 60 chemicals were tested. Some were independently tested two or four times each. In this preliminaly test, pregnant mice were dosed during mid‐pregnancy and were then allowed to deliver litters. Litter size, birth weight, and neonatal growth and survival to postnatal day 3 were recorded as indices of potential developmental toxicity. Results in this assay and conventional mouse teratology tests were generally concordant. Conventional data were available for 14 chemicals (ten teratogens, one fetotoxin, three nonteratogens), of which 11 (nine teratogens, one fetotoxin, one nonteratogen) produced evidence of developmental toxicity. This included conventional data for three chemicals (ethylene glycol, diethylene glycol dimethyl ether, and triethylene glycol dimethyl ether) that were untested before the present study. As high priority candidates for conventional testing on the basis of results here, all were subsequently studied in a standard teratology assay and were confirmed to be teratogenic in mice. Additionally, one of them (ethylene glycol) plus a fourth high priority candidate for conventional study (diethylene glycol monomethyl ether) were subsequently tested in rats and were found to be teratogenic in that species.
Recommendations for the statistical analysis of Chernoff/Kavlock test data
Authors: Burg JeAnne R. , Hardin Bryan D.
Published: February 2006

The statistical analyses of the data related to the teratogenic testing of 60 compounds utilizing the Chernoff/Kavlock test are the basis of this paper. The hypotheses chosen to be tested and the statistical tests utilized to test those hypotheses are discussed. The topics explored are the use of comparisonwise error rate versus experimentwise error rate, the use of nonparametric tests versus more “conventional” parametric tests and the use of “historical data” in assessing and interpreting group difference.
A recommended protocol for the Chernoff/Kavlock preliminary developmental toxicity test and a proposed method for assigning priority scores based on results of that test
Authors: Hardin Bryan D.
Published: February 2006

Recommendations are made for a standard protocol for the Chernoff/Kavlock preliminary developmental toxicity test, and a weighted scoring system is proposed to provide a numerical prioritization. Protocol recommendations include use of pregnant animals for dose‐finding studies and, in the testing phase, treatment on gestation days 6–15 at an overtly maternally toxic dose. This treatment period corresponds to the usual timing of a teratology test and provides an improved data base in the event a conventional test is deemed appropriate. Five indices of potential developmental toxicity are: the proportion of pregnant survivors at term that produces a viable litter (at least one liveborn pup), average litter size and pup weight at birth, and average neonatal survival and body weight gain to 3 days of age. Neither systematic examinations of pups (living or dead) for malformations nor counts of dead pups are considered productive. In the proposed method for point scores, each of the five indices is assigned a point value that varies according to broad classes of maternal toxicity. The maximum total point value is 22. When there is no statistically significant difference between treated and control groups, the assigned value is subtracted from 22. If there is a statistically significant treatment effect, or if statistics cannot be applied, then no points are subtracted. The points remaining after all five indices are considered constitute the priority score, which may range from 0 (low) to 22 (high priority).
Application of the preliminary developmental toxicity screen for chemical hazard identification under the toxic substances control act
Authors: Francis Elaine Z. , Farland William H.
Published: February 2006

The Office of Toxic Substances (OTS) within the U.S. Environmental Protection Agency (EPA) is authorized to carry forth the mandates of the Toxic Substances Control Act (TSCA). Included among the provisions of TSCA are the development of requirements for testing of “new” and “existing” chemicals that may present an unreasonable risk of injury to health or the environment. There are over 63,000 “existing” chemicals on the TSCA inventory, and EPA in recent years has been receiving an average of over 1,300 submissions for “new” chemicals a year. Since it is illogical and unrealistic to expect that all of these chemicals should be subjected to detailed testing for all potential adverse health and environmental effects, OTS views screening assays as highly useful tools to assign priorities to chemicals for further testing according to standard methodologies. The Chernoff/Kavlock assay (preliminary developmental toxicity screen) was specifically developed to address the need for a developmental toxicity assay to prioritize for further testing the large number of “new” and “existing” chemicals. OTS has been involved in seeking the development of data through the preliminary developmental toxicity screen for purposes of validating the screen and to obtain critical data necessary for evaluating chemicals. OTS believes that the screen has a role in the risk assessment process and has developed a testing protocol, which is included along with other OTS test guidelines; has provided internal guidance on when the screen may be recommended; and has discussed how the data may be applied in prioritizing chemicals for further study.
Correlations between embryotoxic and genotoxic effects of phenytoin in mice
Authors: Barcellona Patrizio Scorza , Barale Roberto , Campana Alberto , Zucconi Donatella , Rossi Vilma , Caranti Silvia
Published: February 2006

The anticonvulsant drug phenytoin (DPH) has been suspected to produce embryotoxicity through an arene oxide intermediate. This drug was also found to be a genotoxic agent. These hypotheses were tested in pregnant mice modulating the phases I and II metabolizing enzymes. DPH was studied by assessing embryotoxicity, teratogenicity, and genotoxicity, the latter by the micronucleus test on the polychromatic erythrocytes of dams and fetuses.

DPH embryotoxicity was potentiated by inhibiting both cytochrome P‐450 and epoxide hydrase and decreased by inducing cytochrome P‐450. Equivocal results were obtained by modulating cytochrome P‐448. The main DPH metabolite, p‐hydroyphenytoin (HPPH), was ineffective both per se and after cytochrome induction or epoxide hydrase inhibition.

DPH did not exert genotoxicity on the maternal organism, no matter which modulating agent was used. In the fetus, however, weak genotoxic effects were observed. These effects significantly increased with inhibition of epoxide hydrase; they disappeared with induction of both cytochromes P‐448 and P‐450 or with inhibition of the latter. No genotoxicity was exerted by HPPH, even when the enzymatic pattern was modulated.

It is concluded that the major role in DPH embryotoxicity is played by the unchanged drug, while the presence of the arene oxide is determinant for genotoxic effects.
The potential relationship of maternal toxicity, general stress, and fetal outcome
Authors: Chernoff Neil , Kavlock Robert J. , Beyer Patricia E. , Miller Diane
Published: February 2006

Standard teratology bioassays generally call for a top dose level which is sufficient to induce some form of overt maternal toxicity such as death or weight loss. The presence of such maternal toxicity is often a confound in the interpretation of experimental results, especially at those dose levels producing the toxicity. While the physiological bases for the toxicity vary widely in a compound‐related fashion, one underlying factor that remains constant for most induced toxicity is the presence of generalized stress in the affected animals. Previous studies have indicated that pregnant animals treated acutely with toxic levels of a variety of pharmacologically unrelated chemicals produced litters without a recognizable syndrome of defects, except for an increased incidence of supernumerary ribs (SNR). The present study reports on the effects of immobilization stress on the production of SNR in the Sprague‐Dawley rat and the CD‐1 mouse. Pregnant animals were immobilized in the supine position for 12‐hour periods during the day of greatest sensitivity to SNR production (days 9 and 10 in the mouse and rat, respectively). Animals were killed immediately before term and the fetuses were examined. An increase in SNR was noted in immobilized mice but not rats. These results suggest that such fetal effects may be the result of general agent‐induced maternal stress.
Use of the adult developmental relationship in prescreening for developmental hazards
Authors: Johnson E. Marshall , Christian Mildred S. , Dansky Linda , Gabel B. E. G.
Published: February 2006

The overwhelming majority of chemicals already in commerce or brought into use each year have not been evaluated for their potential to adversely affect in utero development. Data from those that have been evaluated thus far in pregnant laboratory animals establish that most, but not all, were no more hazardous to the conceptus than they were to adult homeostasis. Most did not need standard developmental toxicity testing because avoidance of adult toxic exposure levels would have precluded abnormal in utero development. The six general principles of teratology when modified, expanded, and placed into this type of context of contemporary developmental toxicology allow an updating of the present testing sequence which was devised prior to 1966.

The developmental hazard index (A/D ratio) calculated from the adult and developmental NOELs of standard Segment II evaluations is predicted by in vitro means. This determination, when coupled with adequate considerations of exposure can be used to prioritize chemicals for more elaborate developmental toxicity tests. Those chemicals with large ratios, i.e., disruptive of embryogenesis at treatment levels too low to produce overt effects in the mother and/or with significant concern regarding exposure, can be identified and tested in pregnant laboratory animals as high priority items. Those with low ratios and those for which there is a low level of concern regarding exposure potential also can be identified and are not high priority items for testing in pregnant animals. The proposed tier system establishes priorities of testing based on exposure and the concept of target organ toxicity applied to the embryo. It provides intensive in vivo evaluations of those chemicals for which developmental effects testing is most needed and avoids use of resources and animals for unnecessary testing of agents that do not pose threats to the conceptus.
Benomyl‐induced craniocerebral anomalies in fetuses of adequately nourished and protein‐deprived rats
Authors: Ellis William G. , Semple Janet L. , Hoogenboom Elizabeth R. , Kavlock Robert J. , Zeman Frances J.
Published: February 2006

Benomyl, a benzimidazole fungicide, produced craniocerebral and systemic malformations in fetal rats when administered by gavage in doses of 31.2, 62.5, and 125 mg/kg of maternal body weight on days 7–21 of gestation. Malformations increased in incidence and severity with increasing benomyl dosage and nearly doubled when coupled with a protein‐deficient diet. Protein deficiency alone produced only decreased fetal weight. High benomyl doses produced higher percentages of fetal resorptions and late fetal deaths, and these percentages also increased with protein deficiency. A benomyl dose of 62.5 mg/kg in proteindeficiency dams, the optimal combination for a high incidence of anomalies and low fetal wastage, produced hydrocephalus in 69.4% of fetuses, meningocele in 8.2%, encephalocele in 14.3%, exencephaly in 44.9%, anencephaly in 14.3%, corpus callosum agenesis in 26.5%, periventricular necrosis in 26.5%, and periventricular cellular “overgrowth” in 55.1%. The most common combination of anomalies was hydrocephalus, exencephaly, and periventricular “overgrowth.” Common systemic malformations included cleft palate, micromelia, hydroureter, and misshapen tails. No fetus was entirely normal at the highest benomyl dose.

Benomyl has been shown by others to bind tubulin and inhibit the formation of microtubules that are important in neurulation, mitosis, and cell migration during early brain development. Thus, it is suggested that benomyl, coupled with a protein‐deficient diet, offers a teratogenic model with a spectrum of abnormalities similar to hypervitaminosis A but with a higher yield of specific craniocerebral anomalies.
Dissociation of estrogen‐induced uterine growth and ornithine decarboxylase activity in the postnatal rat
Authors: Sheehan Daniel M. , Branham William S.
Published: February 2006

Estrogens are teratogens and developmental carcinogens in several species. We have used uterine growth to quantitate the potency of three estrogens [estradiol (E2), diethylstilbestrol (DES), ethynylestradiol (EE2)] during four postnatal periods (days 1–5, 10–14, 20–24, and 60–64) in the rat. Alphafetoprotein (AFP), present at high levels in neonatal serum, is thought to regulate estrogen bioavailability. Association constants for DES and EE2 were 2.7% and 4.9% of that for E2 binding to AFP, determined in a batch Sephadex equilibrium binding assay. On days 1–5, DES and EE2 were about 80‐fold more potent than E2 in increasing uterine weight. As AFP levels fell, potency differences between E2 and the synthetic estrogens decreased. In the adult, which essentially lacks AFP, the three estrogens were nearly equipotent. These data are consistent with AFP regulation of estrogen potency. On days 10–14, uterine growth was less sensitive than at other ages to all three estrogens, perhaps related to uterine differentiation and/or the high endogenous serum E2 levels reported at this age.

However, when we examined another uterine estrogen response, ornithine decarboxylase (ODC) induction at 6 h following estrogen injection, all three hormones were about equipotent in both neonatal and adult animals. This apparently AFP‐independent event shows dissociation of ODC induction and uterine growth, which could be due to separate mechanisms for hormone entry to target tissue or subsequent intracellular events.
The covalent binding of 1, 1,2,2‐tetrachloroethane to macromolecules of rat and mouse organs
Authors: Colacci Annamaria , Grilli Sandro , Lattanzi Giovanna , Prodi Giorgio , Turina M. Paola , Forti Giorgio Cantelli , Mazzullo Mario
Published: February 2006

The in vivo interaction of the hepatocarcinogen 1,1,2,2‐tetrachloroethane (1,1,2,2‐TTCE) with DNA, RNA, and proteins of male Wistar rats and BALB/c mice was measured 22 hr after i.p. injection. Covalent binding index (CBI) to liver DNA was about 500 and was comparable to those of carcinogens classified as moderate initiators. It was higher than those of other chloroethanes, even than that of 1,2‐ dichloroethane (1,2‐DCE), a symmetrically substituted haloethane whose genotoxicity has been widely demonstrated.

In in vitro cell‐free systems, 1,1,2,2‐tetrachloroethane was bioactivated by mixed‐function oxidase(s) and glutathione‐S‐transferase(s) (GSH‐T) from microsomal and cytosolic fractions of rat and mouse liver and, to a lesser extent, of mouse lung. The in vitro activation led to formation of reactive species capable of binding to exogenous DNA and to the subcellular constituents of enzymatic fractions.

These data, along with previous literature reports, provide sufficient evidence of 1,1,2,2‐TTCE genotoxicity.
Depression of glutathione in male reproductive tissues and potentiation of EMS‐induced germ cell mutagenesis by L‐buthionine sulfoximine
Authors: Teaf Christopher M. , Bishop Jack B. , Harbison Raymond D.
Published: February 2006

Buthionine sulfoximine (BSO) treatment significantly reduced testicular epididymal and vas deferens glutathione (GSH) levels in rats. Testicular levels of GSH were reduced by 20%, while epididymal GSH levels were reduced by more than 50%. BSO treatment correspondingly enhanced ethyl methanesulfonate (EMS)‐induced dominant lethal mutations. EMS‐induced resorption rates were doubled following BSO treatment. This effect was observed in mating wk 2 and 3 (d 8–19 following treatment), indicating effects on those germ cells which were in late testicular stages or were caput epididymal spermatozoa at the time of EMS treatment. The enhancement of the mutagenic action of EMS by BSO is restricted to the same time period (spermatid‐spermatozoa transition, early epididymal maturation) as maximum sensitivity to the clastogenic action of EMS on male germ cells. The temporal pattern of EMS alkylation of rat spermatozoa correlated with the incidence of EMS‐induced dominant lethal mutations. BSO depresses GSH in the male reproductive tract in a dose‐ and time‐dependent manner. Perturbation of GSH in the male reproductive tract appears to influence chemical‐induced germ cell mutations.
Factors influencing the initiation by gamma rays of hepatocarcinogenesis in the rat
Authors: Kaufmann William K. , MacKenzie Susan A. , Kaufman David G.
Published: February 2006

F344 male rats were irradiated once with 6 G of cesium 137 gamma rays at various times after a two‐thirds partial hepatectomy (PH) and then fed a diet containing the liver tumor promoter phenobarbital to evoke the expression of initiated hepatocytes. Yields of hepatocellular neoplasms were enumerated at 45 weeks after irradiation. Although proliferating hepatocytes in regenerating livers appeared to have increased risk of initiation by gamma rays, there was no apparent variation in risk among groups that were treated at times when hepatocytes were in different portions of the cell cycle (G1, S, G2/M). Gamma irradiation delayed the onsets of DNA synthesis and mitosis by proliferating hepatocytes by 18–20 h. The rate of rejoining of radiation‐induced DNA strand breaks was analyzed in primary cultures of hepatocytes; 98% of the strand breaks were rejoined within 30 min after irradiation. Efficient repair of certain types of radiation‐induced damage to DNA before the damaged DNA is replicated should cause a substantial reduction in the probability of induction of base‐substitution mutations. Hepatocellular islands and neoplasms that were initiated by gamma rays may be derived from proliferating hepatocytes which incurred other radiation‐induced DNA damages such as chromosomal aberrations.
Effect of duration of exposure to benzo(a)pyrene diol‐epoxide on neoplastic transformation, mutagenesis, cytotoxicity, and total covalent binding to DNA of rodent cells
Authors: Krolewski Bozena , Little John B. , Reynolds Richard J.
Published: February 2006

We examined the effect of different durations of exposure (20 sec to 24 hr) to (±) 7‐β,8α‐dihydroxy‐9α, 10α ‐epoxy‐7,8,9,10‐tetrahydrobenzo(a)pyrene (BPDE I) on the induction of transformation in C3H/10T1/2 cells and of mutations to 6‐thioguanine resistance in Chinese hamster ovary cells (CHO), as well as on BPDE I‐DNA binding in these two cell lines. A 20‐sec exposure of the cells to BPDE I was sufficient to induce mutations and morphological transformation in vitro. However, the transformation frequency in CH3 mouse‐embryo‐derived 10T1/2 cells increased twofold and the frequency of mutations in CHO cells sixfold when the exposure time to BPDE I was increased from 20 sec to 8 h. Cytotoxicity increased under similar conditions. A large number of BPDE I‐DNA adducts were formed in both cell lines within the first 15‐min of exposure of the cells to this ultimate carcinogen. The total covalent binding did not increase with longer than 15‐min incubation times. These results suggest that in addition to its covalent binding to DNA, BPDE I may influence other cellular mechanism(s) that are responsible for the initiation of transformation and mutagenesis.
Induction of sister chromatid exchanges by ergot compounds in chinese hamster ovary cells in vitro
Authors: Dighe Rajani , Vaidya V. G.
Published: February 2006

Five ergot alkaloids, d‐lysergic acid derivatives, were tested for the induction of sister chromatid exchange (SCE) frequencies in cultured Chinese hamster ovary cells. The concentrations of the test substances ranged between 10−5 and 10−8 M. Ergotamine, ergonovine, and methylergonovine induced a significantly high number of SCEs at all the concentrations used, while with ergocristine this occurred only at the highest concentration. α‐Ergocryptine failed to induce SCEs at any concentration. It is therefore surmised that ergotamine, ergonovine, and methylergonovine are effective inducers, ergocristine is a weak inducer, and α‐ergocryptine is a noninducer in Chinese hamster ovary cells in vitro.
Development of a metabolic activation system for the frog embryo teratogenesis assay: Xenopus (FETAX)
Authors: Fort Douglas J. , Dawson Douglas A. , Bantle John A.
Published: February 2006

FETAX (frog embryo teratogenesis assay: Xenopus) is a 96‐hr teratogenesis screening assay using embryos of the South African clawed frog, Xenopus laevis. Since Xenopus embryos have limited xenobiotic metabolism through 96 hr of development, we have developed an in vitro metabolic activation system employing Aroclor 1254‐induced rat liver microsomes. By adding an exogenous source of mixed functional oxidase (MFO) activity, we may more accurately assess the teratogenic risk of proteratogenic compounds. Xenopus embryos were cocultured with varying concentrations of cyclophosphamide (CP), Aroclor 1254‐induced microsomal protein, an NADPH‐generating system, and antibiotics in a static renewal fashion for 96 hr. Residual Aroclor 1254 remaining in the microsomes was successfully reduced during purification to levels that had no significant effect on embryo survival and development. The results of three definitive dose‐response tests performed with CP revealed that activation reduced the 96 hr LC50 from 8.0 to 1.4 mg/ml (5.7‐fold). The 96‐hr EC50 (malformation) was reduced from 6.2 to 0.4 mg/ml (15.5‐fold). Activation also increased the types and severity of malformation and reduced embryonic growth. Aroclor 1254‐induced rat liver microsomes may be used as an acceptable in vitro metabolic activation system for FETAX.
Effects of sodium azide on sea urchin embryos and gametes
Authors: Pagano G. , Cipollaro M. , Corsale G. , Esposito A. , Mineo A. , Ragucci E. , Giordano G. G. , Kobayashi N. , Trieff N. M.
Published: February 2006

Sodium azide (SA) was tested on sea urchin embryos and gametes (Paracentrotus lividus). Developing embryos were exposed to SA (10−6 to 10−3 M) up to pluteus larval stage, or for shorter intervals before or after hatching. Developmental defects in SA‐exposed embryos consisted mainly of gut abnormalities, without any detectable differences between pre‐ or post‐hatch‐exposed embryos. SA‐induced damage to gut was exerted during gastrulation, as evident by lectin binding of extracellular matrix. No mitotic damage was observed in SA‐exposed embryos, nor could pH‐related variations be detected in SA‐induced embryotoxicity at pH's ranging from 8 to 6. Concurrently, no effect ensued in the exposure of unfertilized eggs to SA (10−5 to 10−2 M) both in terms of fertilization success and of offspring quality. When sperm were suspended in filtered seawater at pH's ranging from 8 to 6, and SA levels ranging from 10−5 to 10−2 M, fertilization success of SA‐exposed sperm appeared to be modulated by pH, by displaying three distinct dose‐response trends at pH 8, 7, or 6. The consequences of sperm pretreatment on offspring quality failed to show any significant SA‐induced changes on larval malformations or mortality, while confirming the previously reported pH‐induced increase of developmental defects in the offspring of acid‐exposed sperm (Pagano et al.: Teratogenesis Carcinogen Mutagen 5:113–121, 1985).
Effects of in vivo and in vitro exposure to rhodamine dyes on mitochondrial function of mouse embryos
Authors: Ranganathan Sulabha , Hood Ronald D.
Published: February 2006

Cationic rhodamines (Rh 123 and Rh 6G) can cause developmental toxicity in mice and inhibit embryonic mitochondrial respiration following in vivo or in vitro dye exposure. Rh B, a neutral rhodamine, fails to show such effects at comparable doses. To assess effects of rhodamines on development, F0F1ATPase activity and ADP translocation were measured on gestation day (GD) 12 in embryonic and adult mitochondria. ATP synthesis in embryonic mitochondria transplacentally exposed to Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) given to dams by i.p. injection from GD 7 to 10 were inhibited 39% and 49%, respectively. When isolated mitochondria were treated, dose‐dependent inhibition was seen; at 5 μg of dye/mg mitochondrial protein, ATP synthesis was inhibited 65% and 81% by Rh 123 and Rh 6G, respectively. When F0F1ATPase activity was assessed, in vitro Rh 123 and Rh 6G exposures at levels up to 8 μg/mg mitochondrial protein resulted in enzyme inhibition, but at 10 μg/mg, ATPase activity was stimulated. Uncoupler‐stimulated ATPase activity was also inhibited. ADP translocation was decreased by 19.1% and 37.7% by Rh 123 and Rh 6G, respectively, at dye concentrations of 20 μg/mg. Results of in vitro exposure of maternal liver mitochondria were similar to those for embryonic mitochondria, whereas liver from dams exposed in vivo on GD 7–10 was unaffected on GD 12. In vivo or in vitro treatment with Rh B did not affect any embryonic or maternal parameters. Such results support the hypothesis that inhibition of mitochondrial energy metabolism is a mechanism for the developmental toxicity of cationic rhodamines.
Limb bud cell culture for in vitro teratogen screening: Validation of an improved assessment method using 51 compounds
Authors: Renault J.‐Y. , Melcion C. , Cordier A.
Published: February 2006

Rat embryo limb bud cells multiply and undergo chondrogenesis in micromass culture. Teratogenic agents are identified from their inhibition of chondrogenesis, which is quantified by determination of cartilaginous foci number or proteoglycan production. In other in vitro systems, the detection is based on their ability to affect cell proliferation. So far, these methods have failed to distinguish among true inhibition of differentiation, inhibition of cell proliferation, and nonspecific cytotoxicity. The improved technique involves simultaneous measurement of cartilage synthesis and cell multiplication. Differentiation was evaluated by measurement, using an Artek Counter, of nodule areas after Alcian blue staining and proliferation by spectrophotometric quantification of Crystal‐Violet bound to micromass cells. Using this method, retinoic acid was shown to inhibit chondrogenesis without affecting cell multiplication, whereas 6‐aminonicotinamide preferentially inhibited cell multiplication without affecting nodule size. Doxylamine (succinate), a known nonteratogen, induced inhibition of chondrogenesis, but with a parallel inhibition of cell multiplication, reflecting a nonspecific toxic effect. This improvement increases the specificity of the micromass culture test. Validation was performed using 51 compounds. Compounds were classified according to their inhibitory activity and their active concentration. The sensitivity of the test was 61%; the specificity, 100%; and the final accuracy, 75%. The method is fully miniaturised, automated, and computerised, allowing numerous compounds to be rapidly tested at very low cost.
Mutagenic lipid peroxides from edible oils
Authors: Kensese Sontin Mossanda , Teng Jon I. , Smith Leland L.
Published: February 2006

Weak mutagenic activity was detected in several commercially available edible palm and corn oils using liquid incubation bioassays with Salmonella typhimurium TA1537. Chromatographic fractionation of unrefined palm oil established that mutagenic activity was present in three fractions that also contained fatty acyl hydroperoxides. Similar weak mutagenic activity was also demonstrated for linoleic and linolenic acid hydroperoxides. In all cases, the mutagenicity was abolished by exogenous catalase, implying that the observed activity was moderated by hydrogen peroxide.
Cell cycle alterations and cell death in cyclophosphamide teratogenesis
Authors: Chernoff Neil , Rogers John M. , Alles Ajit J. , Zucker Robert M. , Elstein Kenneth H. , Massaro Edward J. , Sulik Kathleen K.
Published: February 2006

Litters of pregnant mice treated with cyclophosphamide (CP) exhibit malformations of the limbs ranging from oligodactyly to amelia. Previous studies have indicated that cell death occurs in limb buds shortly after maternal exposure. We have investigated the relationship of cell death, cell cycle perturbation, and embryo/fetal toxicity in the mouse using vital staining and flow cytometry (FCM). CP (20, 30, and 40 mg/kg) was investigated via intraperitoneal administration to Swiss‐Webster mice on day 10 of gestation. At 4, 8, or 28 hours later, embryos were removed. Cell death was identified with Nile blue sulphate (NBS). Two embryos per litter were stained with NBS, and the remaining embryos were frozen at – 70°C prior to FCM analysis. After thawing, the forelimb buds were removed for the isolation of nuclei. Tissues were dissociated through a wire mesh followed by cytolysis with 0.1% nonidet P‐40 in PBS with 0.5 mg/ml RNase. Nuclei were stained with the fluorescent nucleic acid probe propidium iodide and analyzed (10,000 nuclei per sample) for propidium iodide fluorescence by FCM. NBS revealed a dose‐related increase in cell death by 8 hours after dosing. CP‐induced cell death was greatest in areas of rapid cell proliferation (DNA synthesis). FCM analysis revealed retardation of progression through the S‐phase of the cell cycle by 4 hours post‐exposure at all doses. This retardation occurred earlier in S‐phase with increasing dose and persisted through 8 hours. At 28 hours, cell cycle histograms were normal in the low‐dose embryos, but remained perturbed in the intermediate‐ and high‐dose embryos. On day 17 of gestation, the last group of dams was killed. A high incidence of fetal malformations, including limb defects, occurred at the 20 mg/kg dose, and fetal mortality was observed at 30 and 40 mg/kg. The pattern and magnitude of cell deth corelated with cell cycle perturbation and fetal toxicity at term, suggesting a relationship between cell cycle perturbation, cell death, and malformations produced by CP.
Biochemical studies of promoters of carcinogenesis in rat liver
Authors: Kitchin Kirk T. , Brown Janice L.
Published: February 2006

Adult female rats were orally dosed with 1/5 to 3/5 the published LD50 of either promoters or putative promoters of carcinogenesis [hexachlorobenzene (HCB), α‐hexachlorocyclohexane (α‐HCH), kepone and toxaphene] or noncarcinogens [coumaphos, EDTA, caprolactam, 8‐hydroxyquinoline, titanium (IV) oxide, sodium diethyldithiocarbamate (DEDTC), and sucrose] at 21 and 4 h before sacrifice. The promoters selected in this study were all of the halogenated hydrocarbon class. At doses of 1/5 to 3/5 the LD50, all four promoters or putative promoters induced rat hepatic ODC activity. The seven noncarcinogens produced several biochemical effects at doses of 1/5 the LD50: increased serum alanine aminotransferase activity (SGPT) (caprolactam and DEDTC), decreased hepatic cytochrome P‐450 content (DEDTC), and increased hepatic ODC activity (8‐hydroxyquinoline and DEDTC). None of the seven noncarcinogens caused hepatic DNA damage or coordinate induction of hepatic ODC and cytochrome P‐450. The results support the interpretation that several of these biochemical parameters are useful in distinguishing potential tumor promoters and noncarcinogens.
Genotoxic effects of fly ash in bacteria, mammalian cells and animals
Authors: Morris D. L. , Connor T. H. , Harper B. L. , Ward J. B. , Legator M. S.
Published: February 2006

The increasing use of fossil fuels has raised concerns about possible deleterious health effects of the final combustion product, fly ash. Seven ash samples from coal sources obtained from Battelle Columbus Laboratories were evaluated in the Salmonella/mammalian microsome mutagenicity assay to determine their mutagenic potential. While dimethyl sulfoxide extracts of five samples showed no mutagenicity, sample 102 caused an increase in the number of revertants per plate over controls in TA100 and TA98 with activation by liver homogenate (2‐fold and 2.4‐fold, respectively) and without (2‐fold and 6‐fold). This ash was thus evaluated in whole animal studies. Animals treated by inhalation or oral gavage were assayed for the presence of mutagens in the urine, micronuclei in polychromatic erythrocytes, and chromosomal aberrations in metaphase bone marrow cells. Those animals treated by inhalation were also examined for local damage in the lung. The assay for mutagens in the urine was negative as shown by the Ames assay with TA100 and TA98 and there was no increase in micronuclei or in metaphase aberrations. Histological sections from the animals treated by inhalation did not show the presence of particles, macrophage infiltrations and generalized lung damage. We tested the same fly ash with an in vitro cell transformation assay with the cell line Balb/c 3T3 subclone A31‐1‐13. Although there was not an increase in Type III foci, there was a dose‐dependent increase of Type II foci in the treated cells over the controls. In one assay, there was approximately a 14‐fold increase in Type II foci in the highest dose (2 mg/ml) compared to the solvent control. One other ash sample induced cell transformation without being markedly cytotoxic, while a third sample was highly toxic but did not induce transformation.
Tetrachloroethane, pentachloroethane, and hexachloroethane: Genetic and biochemical studies
Authors: Bronzetti G. , Morichetti E. , Carratore R. Del , Rosellini D. , Paolini M. , Cantelli‐Forti G. , Grilli S. , Vellosi R.
Published: February 2006

Tetrachloroethane (TTCE), pentachloroethane (PCE), and hexachloroethane (HCE) were tested in diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension test with and without mammalian metabolic activation (S9). TTCE, PCE, and HCE gave positive results on cells harvested from logarithmic growth phase; only PCE induced a significant increase (P ≤ .01) of mitotic gene conversion and point reverse mutation on cells from stationary growth phase with metabolic activation (S9).

The in vivo effects on cytochrome P450 content (cyt. P450), pentoxyresorufin O‐dealkylase (P450‐like, class IIB, PROD), and ethoxy‐resorufin O‐deethylase (P448‐like, class IA, EROD) activities were examined in hepatic microsomes from mice 24 h after acute intoxication. All the halogenated hydrocarbons displayed a marked toxic effect as shown by the significant decrease in cyt. P450 levels (maximum of 76% decrease, with TTCE 753.2 mg/kg) and EROD (maximum of 69% decrease, with PCE 925.4 mg/kg), and to a lesser extent in PROD (maximum of 52.4% decrease, with HCE 3150 mg/kg). Although a general decrease of P450 functions was observed, the toxic effects of TTCE and PCE seem to be preferentially related to P448 forms.
Noncorrelative c‐myc and ras oncogene expression in squamous cell carcinoma cells with tumorigenic potential
Authors: Shuler Charles , Kurian Ponnamma , French Bernard T. , Noyes Inge , Sital Nirander , Hollering Jens , Trewyn Ronald W. , Schuller David , Milo George E.
Published: February 2006

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface‐associated antigens. Increased levels of H‐ras mRNA and p21 protein were present in six tumors, but enhanced c‐myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen‐positive cells was not uniform throughout the tumor.

Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC‐83‐01‐82, isolated by passage through soft agar, was treated with 50 μg/ml of methyl methane sulfonate (MMS). These MMS‐converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c‐myc and H‐ras mRNA expression. Serial passage of the MMS‐converted tumors in vivo was accompanied by consistent enhanced c‐myc expression. However, the levels of H‐ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c‐myc and H‐ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC‐83‐01‐82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c‐myc and ras gene expression.
New structural concepts for predicting carcinogenicity in rodents: An artificial intelligence approach
Authors: Rosenkranz Herbert S. , Klopman Gilles
Published: February 2006

The Computer Automated Structure Evaluation (CASE) method for studying structure‐activity relationships has been applied to a data base of rodent carcinogens. It has been demonstrated that CASE is able to identify determinants embedded in the molecular structure which, with a high probability, predict rodent carcinogenicity. CASE has also identified determinants associated with the activity of non‐genotoxic carcinogens, thereby suggesting that there is a structural commonality in the activity of these molecules. The present study reveals that there are “universal” as well as species‐specific structural determinants of carcinogenicity. CASE was able to predict the carcinogenicity in rodents of certain endogenous pesticides in edible plants.
Tumor promotion: Models and assay systems
Authors: JamesFitzgerald D. , Yamasaki Hiroshi
Published: February 2006

Tumor promotion is defined operationally from two‐stage models of experimental carcinogenesis. It is, therefore, in a strict sense, possible to identify tumor promoters only from such models. The development and use of in vitro two‐stage cell transformation assays was a logical extension toward in vitro short‐term testing for tumor promoters. Another approach is to apply mechanistic knowledge of the tumor promotion process in developing end points for such assays. In this context, we have been examining the role of blocked gap‐junctional intercellular communication (GJIC) in tumor promotion, using in vitro and in vivo systems. Many promoters have been shown to block GJIC in vitro; our studies support the idea that inhibition of GJIC does play an important role in the promotion stage of BALB/c 3T3 cell transformation. In animal studies, we have shown that the rat liver tumor promoter phenobarbital can decrease the level of expression of the 32 Kd gap junction protein gene specifically in liver upon systemic exposure in rats. Further examination of the role of GJIC in tumor promotion is indeed warranted. Also, deployment of in vitro GJIC and transformation assay systems should provide useful short‐term tests for detecting tumor promoting activity of environmental chemicals.
Molecular analysis of reactive oxygen‐species‐induced mammalian gene mutation
Authors: Hsie Abraham W. , Xu Zhidong , Yu Yongjia , Sognier Marguerite A. , Hrelia Patrizia
Published: February 2006

We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1‐BH4 and its transformant, AS52. The AS52 cells lack the normal X‐linked mammalian hypoxanthine‐guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine‐guanine phosphoribosyltransferase (gpt) gene. We found that X‐rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1‐BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen‐producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi‐locus deletion mutants in AS52 cells than in K1‐BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.