Potent Rodent Research Articles

Anthranoid laxative abuse--a risk for colorectal cancer?

Anthranoid-containing laxatives--aloe, cascara, frangula, and rheum--may play a role in colorectal cancer. This risk is particularly important in view of the wide abuse of self administered laxatives for chronic constipation. There are data on the genotoxic potential of anthranoids and there is evidence of a tumourigenic potential in rodents. A case report and clinical-epidemiological studies have evaluated the cancer risk in patients who have abused anthranoid laxatives over a long period. Pseudomelanosis coli is a reliable parameter of chronic laxative abuse (> 9-12 months) and is specific for anthranoid drugs. In a retrospective study of 3049 patients who underwent diagnostic colorectal endoscopy the incidence of pseudomelanosis coli was 3.13% in patients without pathological changes. In those with colorectal adenomas, the incidence increased to 8.64% (p < 0.01), and in those with colorectal carcinomas it was 3.29%. This lower rate was probably caused by incomplete documentation of pseudomelanosis coli in those with carcinoma. In a prospective study of 1095 patients, the incidence of pseudomelanosis coli was 6.9% for patients with no abnormality seen on endoscopy, 9.8% (p = 0.068) for patients with adenomas, and 18.6% for patients with colorectal carcinomas. From these data a relative risk of 3.04 (1.18, 4.90; 95% confidence interval) can be calculated for colorectal cancer as a result of anthranoid laxative abuse.

Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines

Aromatic amines have long been recognized as animal and human carcinogens. Recently heterocyclic aromatic amines (thermic amines) have been found in small amounts in cooked foods, primarily meats, and have proven to be potent mutagens and rodent carcinogens. Availability of quantitative databases for mutagenic potency in Salmonella and for carcinogenic potency in rodents has made possible a study of ten heterocyclic thermic amines and 24 aromatic amines. Potencies on mutagenic and carcinogenic scales were significantly correlated. By multiple linear regression analysis and multivariate analysis of variance, two descriptive structural factors were found to modulate the two modes of biological response. These factors were number of rings and methyl substitution at carbon atoms. The quantitative correlation between mutagenic and carcinogenic potencies and the modulating structural factors suggest a significant similarity of molecular mechanisms and support the utility of the short-term bacterial assay in evaluating hazard levels.

The non-genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-cresidine

The two potent rodent bladder carcinogens o-anisidine and p-cresidine, and the structurally related non-carcinogen 2,4-dimethoxyaniline, have been extensively evaluated for genotoxicity to rodents and found to be inactive. Most data were generated on o-anisidine, an agent that is also only marginally genotoxic in vitro. The two carcinogens induced methaemoglobinaemia in rodents indicating that the chemicals are absorbed and metabolically oxidized. Despite their total lack of genotoxicity in vivo, the two carcinogens have the hall-marks of being genotoxic carcinogens given that most test animals of both sexes of B6C3F1 mice and F344 rats are reported to have succumbed rapidly to malignant bladder cancer. No reasons for this dramatic conflict of test data are so far apparent. The experiments described involve, in one or other combination, 2 strains of mice (including B6C3F1) and 4 strains of rat (including F344), the use of oral and i.p routes of exposure and observations made after 1, 3 or 6 doses of test chemical. 6 tissues (including the rat bladder) were assayed using 3 genetic endpoints (unscheduled DNA synthesis, DNA single-strand breaks and micronuclei induction). Aroclor-induced rats were employed in one set of experiments with o-anisidine. In the case of one set of mouse bone-marrow micronucleus experiments the same batch of the 3 chemicals as used in the cancer bioassays, and the same strain of mouse, were used. Possible further experiments and the implications of these findings are discussed.

Inhalation toxicology of isoprene in F344 rats and B6C3F1 mice following two-week exposures.

Isoprene (2-methyl-1,3-butadiene) was selected for toxicologic evaluations because of its structural similarity to 1,3-butadiene, a potent rodent carcinogen. Two-week inhalation toxicology studies of isoprene were conducted in F344 rats and B6C3F1 mice at exposure concentrations of 0, 438, 875, 1750, 3500, or 7000 ppm. For rats, there were no chemically related changes in survival, body weight gain, clinical signs, hematologic or clinical chemistry parameters, or gross or microscopic lesions. Exposure of mice to isoprene did not produce mortalities and only caused a decrease in body weight gain for male mice in the 7000 ppm exposure group; however, hematologic changes and microscopic lesions including testicular atrophy, olfactory epithelial degeneration, and forestomach epithelial hyperplasia were observed in isoprene-exposed mice. Similar toxicologic effects have been previously observed in B6C3F1 mice exposed to 1,3-butadiene. A species difference in susceptibility between rats and mice exposed to isoprene was evident in these short-term exposure studies.

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents

A quantitative correlation between carcinogeneity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodens and mutagenic (M), recombinogenic (R) and SOS-inducting (I) potencies in a bacterial test ( E. coli multitest) for 9 monofunctional alkylating agents: N-methylurethane, N-nitroso- N-ethylrurea, epichlorohydrin, N-nitroso- N-methylurea, N-nitroso- N-methyl- N′- nitroguanidine, methyl methanesulfonate, diethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombigenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.

Quantitative relationship between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. A study of 11 direct-acting monofunctional alkylating agents

This work attempted to derive a quantitative relationship between mutagenicity and carcinogenicity by examining the association between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance. Lethality was measured at equivalent experimental conditions to those of mutant yield by using a mixed population of a pair of isogenic strains distinguished by their differential nutritional requirements. The study was carried out with a group of 11 direct-acting monofunctional alkylating agents, which failed to show any quantitative correlation in the histidine reverse-mutation test. Our data suggest that the mutagenic efficiency of the compounds is directly proportional to the magnitude of the maximum yield of L-arabinose resistance mutants and inversely proportional to the dose and the number of lethal hits at which the maximum yield occurs. A highly significant correlation (r10 = 0.86, P less than 0.01) was found between the mutagenic efficiencies of the compounds in the Ara test and their carcinogenic potencies in rodents, expressed as TD50 ('tumor dose' 50) values. The result suggests that the Ara forward-mutation test of S. typhimurium might be capable of reflecting the relative potency of animal carcinogens, at least when confined to particular chemical classes. A more generic and definitive conclusion about the predictive value of the Ara test would require this analysis to be extended to other types of genotoxic carcinogens.

Nucleophilic selectivity of alkylating agents and their hypermutability in Drosophila as predictors of carcinognic potency in rodents

The nucleophilic selectivity (Swain-Scott s constant or initial 7-alkylguanine/O6-alkylguanine ratio in DNA) of 60 alkylating agents, mostly monofunctional or cross-linking was compared to their carcinogenic potency in rodents (median TD50 estimates) and to two genotoxicity indices in Drosophila: (i) hypermutability, measured by the increased frequency of induced sex-linked recessive lethal mutations (SLRL) in a strain defective in DNA excision repair (exr-), as compared to the wild-type (exr+); (ii) relative clastogenic efficiency, expressed by the ratio of chromosomal aberrations (ring-X loss) to SLRL determined in the exr+ strain. For a subset of direct-acting, monofunctional alkylating agents, nucleophilic selectivity and TD50 values or hypermutability indices were linearly correlated. In addition, the hypermutability indices in Drosophila by methylating or ethylating procarcinogens were similar to the corresponding values of their ultimate metabolites. In contrast, cross-linking agents, including antitumour drugs, did not show these positive correlations. The relative clastogenic efficiencies in Drosophila of 26 direct-acting, alkylating carcinogens increased with both their cross-linking activity and nucleophilic selectivity. By analyzing mutational spectra in Drosophila induced in the vermilion gene by four monofunctional alkylating agents with contrasting s values, critical DNA lesions, i.e. type of base pair substitution mutations, deletions, insertions, involved in genotoxicity were pinpointed. Thus, these multi-endpoint analyses should, as a new approach, assist in the quantitative risk evaluation of genotoxic agents.

Contamination of urban air in a snowy district with 1-nitropyrene and particulate matter during the winter.

Nitroarenes, potent rodent carcinogens, have attracted much attention as environmental contaminants. Recently, simultaneous exposure to dust have been found to potentiate tumorigenicity of nitroarenes in rats (14). We hypothesized that inhabitants of northwestern Japan may be exposed to such substances in winter. First, we sought to develop a sensitive yet time saving method to determine nitroarene concentrations in airborne particulate matter. The method developed used a high-performance liquid chromatograph equipped with a fluorescence detector. By on-column catalytic reduction, using silica coated with platinum and rhodium, 1-nitropyrene (1-NP), 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene were effectively reduced to their amino derivatives which were easily detectable with the fluorescence detector. The lowest detectable limits found using the present method were on the order of picograms for individual compounds. Subsequently, we sought to determine the concentrations of nitroarenes using the new method on airborne particulate matter during the period from November to April. Samples were collected at sampling sites on road with high-volume air samplers for 24 hours or 72 hours in four different rural areas in Iwate Prefecture. At one location in the city of Morioka, samples were collected at the height of 25 m from the ground. Among the four nitropyrenes mentioned above, only 1-NP was detected in the samples. The concentrations of 1-NP and airborne particulates changed significantly in all examined areas in parallel with the rise and fall of the frequencies of studded tire use. The highest 1-NP and airborne particulate concentrations were 1.210 pg/m3 and 1,291 micrograms/m3, respectively, being recorded at the busiest road in Kitakami. In contrast, the lowest values were 38 to 66 pg/m3 for 1-NP and 54 to 94 micrograms/m3 for airborne particulates in Morioka, being recorded in November and April, respectively. The mutagenicities of extracts of airborne particulate matter showed similar trends. Thus, exposure situations with high concentrations of dust and 1-NP were confirmed to occur in Iwate. Finally, the risk posed by 1-NP in airborne particulate matter was estimated using the equation proposed by Cuddihy and McClellan (37) and was found to range from 3.12 x 10(-2) to 24.2 x 10(-2) lung cancer/year/10(6) people. Although the estimated risk due to 1-NP is relatively small, further surveillance is highly recommended in those areas since heavy dust exposure also occurs there.

Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph Series.

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans, an analysis was performed on agents that were evaluated in Supplements 6 and 7 to the IARC Monographs for their carcinogenic effects in humans and animals and for the activity in short-term genotoxicity tests. The prevalence of genotoxic carcinogens on four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and on agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 149) was determined. A high prevalence in the order of 80 to 90% of genotoxic carcinogens was found in each of the groups 1, 2A and 2B, which were also shown to be multi-species/multi-tissues carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and 3. The results of this analysis indicate that (a) an agent with unknown carcinogenic potential showing sufficient evidence of activity in in vitro/in vivo genotoxicity assays (involving as endpoints DNA damage and chromosomal/mutational damage) may represent a hazard to humans; and b) an agent showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity by rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens. Overall, this analysis implies that genotoxic carcinogens add more to the cancer burden in man than non-genotoxic carcinogens. Thus, identification of such genotoxic carcinogens and subsequent lowering of exposure will remain the main goal for primary cancer prevention in man.

Occurrence and relevance of chemically induced benign neoplasms in long-term carcinogenicity studies.

Recent carcinogenicity studies conducted and evaluated by the National Toxicology Program/National Institute of Environmental Health Sciences were examined to determine the frequency of chemically increased incidences of neoplasia. Many of the chemicals originally selected for study were chosen because of an a priori suggestion that they might be carcinogens. Of the 143 chemical studies evaluated, usually involving male and female rats and mice, 42 (29%) did not induce any neoplasms, 20 (14%) gave marginal or equivocal neoplastic responses, and 81 (57%) showed positive neoplastic responses in one or more of the 524 species-gender experiments. Of these 81 positive studies, 60 (74%) were considered positive based on malignant neoplasia, 16 (20%) were positive due primarily to benign neoplasia, but had supporting evidence of malignant neoplasia in the same organ/tissue, and 5 (6%) were positive based only on benign neoplasia. These five chemicals are a) allyl isothiocyanate (transitional cell papillomas of the urinary bladder in male rats), b) 2-amino-4-nitrophenol (tubular cell adenomas of the kidney in male rats), c) asbestos intermediate range chrysotile (adenomatous polyps of the large intestine in male rats), d) decabromodiphenyl oxide (neoplastic nodules of the liver in male and female rats), and e) nitrofurazone (fibroadenomas of the mammary gland in female rats and benign mixed tumors and granulosa cell tumors of the ovary in female mice). For all but one of these lesions (mammary gland), the occurrence in historic controls is low. Thus, only 5 of the 143 chemicals studied (3.5%) induced benign neoplasia alone, and those observed benign neoplasms are known to progress to malignancy. Accordingly, we consider chemically induced benign neoplasia to be an important indicator of a chemical's carcinogenic potential in rodents, and believe it should continue to be made an integral part of the overall weight-of-the-evidence evaluation process for identifying potential human health hazards.

Age-Related Alterations in Pulsatile Luteinizing Hormone Release: Effects of Long-Term Ovariectomy, Repeated Pregnancies and Naloxone1

Aging of the female reproductive system may be regulated by changes at the hypothalamic, pituitary, and ovarian levels. Long-term ovariectomy (LT-OVX) and/or multiple pregnancies delay age-related deterioration of several parameters of reproductive potential in rodents. We tested whether long-term suppression of cyclic ovarian hormone release that is normally associated with the 4- to 5-day estrous cycle decelerates age-related decreases in the frequency of luteinizing hormone (LH) pulses to assess whether hormonal milieu influences the rate of aging of the pulse generator. We determined the percentage of rats exhibiting pulsatile LH secretion, mean LH levels, and amplitude and frequency of LH pulses in seven groups of ovariectomized (OVX) rats. Young (3-4 mo), middle-aged (8-10 mo), and old (18-22 mo) virgin rats, ovariectomized 4 wk (4WK-OVX) prior to experimentation, were used to determine the effect of age. The effect of long-term ovarian hormone deprivation was tested by ovariectomizing rats at 2-3 mo of age and using them when they were middle-aged (8-10 months) or old (18-22 mo). The effect of deprivation of cyclic increases in ovarian hormones associated with repeated estrous cycles was tested by using retired breeder (RB) rats that had been ovariectomized 4 wk prior to experimentation. Each rat was implanted with a right atrial cannula and bled the next day at 10-min intervals for 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenic potency in rodents versus genotoxic potency in E. coli: a correlation analysis for bifunctional alkylating agents

The mutagenic (M), recombinagenic (R) and SOS inducing (I) potencies of 6 bifunctional directly acting alkylating agents (mitomycin C, thiotepa, chlorambucil, nitrogen mustard, bis(2-chloroethyl)ether and bis(2-chloroethyl)nitrosourea) were measured in an E. coli test system ( E. coli multitest) as the integral under the yield-dose curve obtained for each event. This potency crresponds to the cumulative yield of the affected cell population over the entire effective dose range of the chemical treatment. A weak mutagenic activity was detected only for mitomycin C and thiotepa. Except for bis(2-chloroethyl)ether, all agents were recombinagenic and SOS inducing. When the 3 genotoxic potencies (M, R and I) of these bifunctional alkylating agents were correlated, separately or in combination, with the respective carcinogenic potencies in rodents, a highly significant correlation was obtained with both the recombinagenic and SOS inducing potencies.

Evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate

The carcinogenic potential of tilidine fumarate, a synthetic analgesic, was studied for 80 and 104 weeks in mice and rats, respectively. Groups of 50 albino CF1 mice and 65 albino Wistar rats of each sex received tilidine fumarate-lactose blend (1:1) at doses of 100, 40 and 16 mg/kg. The control groups consisted of 100 mice and 115 rats of each sex and received the lactose vehicle only. Treatment-related non-neoplastic changes consisted of reversible, increased cytoplasmic eosinophilia of hepatocytes in high and mid dose rats corresponding to areas of proliferating smooth endoplasmic reticulum; and an increased in high dose rats of proliferative or cystic lesions of the biliary epithelium. Adequate survival rates allowed stringent statistical analysis of neoplasia. Tilidine did not evoke increased tumor incidences or changes in teh average latency or onset of tumors in either species. The most frequent tumors represented spontaneous neoplasia characteristic of historical background incidence in these strains. In mice, the only statistically significant ( P < 0.01) variation in tumor incidence was an increased rate of lung alveologenic adenocarcinomas in females at 100 mg/kg (24%), compared with the concurrent untreated controls (10%), but without a statistically significant difference from historical control data (27%). Female rats given 100 mg/kg showed statistically significant ( P < 0.01) decreased incidences of mammary fibroadenoma and pituitary adenoma. From these data, it was concluded that the synthetic analgesic tilidine does not possess tumorigenic potential in rodents.

Effect of long-term metronidazole (MTZ) therapy on experimental colon cancer in rats

Current reports suggest a beneficial effect of long-term metronidazole (MTZ) therapy in Crohn's disease. Since Crohn's disease is associated with a higher risk of bowel cancer and long-term MTZ has been shown to have a tumorigenic potential in rodents and a cocarcinogenic effect in experimental colon cancer, more studies are required to explore this area. Eighty-one rats were divided into four groups. Group A served as a control, groups B and C were given MTZ in their food (50 mg/kg/day). In groups C and D, a 3-cm colonic segment was isolated and brought out as a blind loop fistula. All animals received 20 weekly sc doses of 1,2-dimethylhydrazine (DMH) and were killed 25 weeks after the first injection. The mean number of colon tumors per animal (±SEM) in MTZ groups B (1.65 ± 0.29) and C (2.57 ± 0.38) were higher than A (1.44 ± 0.3) and D (1.18 ± 0.21), but the increase was only significant for group C over groups A and D ( P < 0.05) and group B ( P = 0.06). The mean number of tumors per animal in the isolated loop of group C (0.95 ± 0.28) was similar to group D (0.68 ± 0.16) P = 0.41, but the mean number of tumors in the functioning colon of group C (1.62 ± 0.25) was higher than group D (0.5 ± 0.12) P < 0.001. These findings suggest that (1) long-term MTZ increased the number of colon tumors per rat in the DMH model but a statistical significance ( P < 0.05) was only noted in the MTZ and surgery group. (2) Surgery alone did not increase the number of tumors. (3) This effect of MTZ is dependent on the presence of the fecal stream, since there was no significant difference between the number of tumors in the empty loops of MTZ and non-MTZ groups.

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O6: 7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents. There was a positive relationship between carcinogenicity and the initial ratios of 7-: O 6-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O 6 (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.

Discriminative stimulus effects of N-substituted analogs of phencyclidine in rhesus monkeys

In daily sessions of a two-lever, discrete-trial, food-reinforced procedure, rhesus monkeys were trained to discriminate between subcutaneous injections of ketamine (1.0 or 1.8 mg/kg) and control injections. In tests of stimulus generalization, cumulative doses of drugs were administered in single sessions and either control- or ketamine-appropriate responding produced food. Ketamine (1.8 and 3.2 mg/kg) and phencyclidine (0.32 mg/kg) produced an average of more than 90% ketamine-appropriate responding. In contrast, d-amphetamine, atropine, chlorpromazine, codeine, diazepam and quipazine, tested at doses up to and including those that markedly reduced response rates, produced exclusively control-appropriate responding. Dose-related ketamine-appropriate responding was produced by each of ten 1-phenylcyclohexylamines, the potencies of which varied with the length, electronegativity, and number of N-alkyl chains present. The most potent analog of phencyclidine, N-ethyl-1-phenylcyclohexylamine, was approximately equipotent with phencyclidine. These data are consistent with previous reports that the discriminative stimulus effects produced by phencyclidine are representative of a unique class of drugs, and that alkyl substitutions in the region of the piperidine ring alter the potency, but not the characteristic pharmacological activity, of the resulting analogs. The potencies of some of these analogs compared to phencyclidine in rhesus monkeys, however, differed from their relative potencies in rodents. Thus, there appear to be species differences in the role of the nitrogen pharmacophore of these compounds in producing phencyclidine-like behavioral effects.

Structure of two adenovirus type 12 transforming polypeptides and their evolutionary implications.

The human adenoviruses are classified according to their nucleotide sequence homology and their oncogenic potential in rodents. The left-hand end of the genome of the adenovirus types 5 (ad5), 7 (ad7) and 12 (ad12), which are respectively, non-oncogenic (subgroup C), weakly oncogenic (subgroup B) and highly oncogenic (subgroup A), contains all the genetic information needed to induce and maintain the transformed phenotype. This part of the genome contains the early transcription unit designated E1 which is subdivided in two transcription units E1A and E1B. Two spliced mRNAs are transcribed from the E1A region which codes for several phosphorylated polypeptides. These polypeptides play a key role by controlling the expression of the other early transcription units. The major role of region E1A in adenovirus cell transformation might not be activate the true transforming genes of the region E1B. An additional role probably consists of the activation of some cellular genes as a restriction fragment containing this region can immortalize rodent cells in vitro. An important question is why some adenoviruses are oncogenic and others are not. We report here differences in the structures of the E1A polypeptides from ad7 and ad12, compared to ad5, which may partially account for their differing oncogenicity.

Effects of Four Years Storage on Germination of R-55 Repellent-Treated White Spruce Seeds

The need for a more effective coating formulation to protect white spruce seeds from rodent depredation led to experimentation using a potent rodent repellent, R-55. Germination tests were conducted at two- to three-month intervals over a period of four years to determine possible phytotoxic effects of the R-55 coating when seeds were returned to cold storage. Germination of treated seeds declined only moderately over this period.

Effect of storage on germination of R-55 repellent-treated seed of white spruce

Determination of seed fate by radio-tracer methods have indicated seed losses to small mammals in Alberta could be as high as 50% during some years even when the widely accepted formulation of aluminum powder-endrin-arasan-latex had been applied to protect the seed supply in direct seeding operations. The need for a better seed coating formulation led to the development and evaluation of a potent rodent repellent, R-55, to protect spruce seeds from rodent damage. As part of the evaluation of this repellent, white spruce seeds were coated with a 4:1 (weight ratio) mixture R-55/graphite and latex. Treated and non-treated (controls) seed lots were stored in a refrigerator for a 2-year period during which eight germination tests were conducted at 2–3 month intervals to test seed viability. Except for one slight mid-winter decline in germination corresponding to a possible seasonal response of the seeds, no significant decline in germination resulted with storage up to 2 years duration. Studies with other coniferous seeds suggest considerable variability in seed species in response to the R-55/graphite coating and to storage of the treated seed.

The dysmorphogenic potential of phenytoin: experimental observations.

Phenytoin, a drug frequently used in the treatment of pregnant epileptic women, has been shown to have an embryotoxic and dysmorphogenic potential in rodents. As in children born to epileptic women, closure of the palate was found to be impaired in mice. In addition, characteristic anomalies such as necrosis, leg anomalies and malformations of the gonads were also observed. In the rat, only a small number of gross malformations were seen, but 20 to 30 % of the surviving fetuses had hydronephrosis. Analysis of recent clinical reports has revealed a good correlation between the experimental findings and the high incidence of congenital malformations in epileptics who received anticonvulsant therapy during pregnancy. The frequency of congenital malformations in children born to treated mothers has been shown to be 2 to 3 times higher than in children born to normal mothers. It seems therefore, that phenytoin is likely to have a dysmorphogenic potential and should be avoided in pregnant epileptic women. The mechanism of the embryotoxic and dysmorphogenic action of phenytoin is still unknown. Attempts to prevent the embryotoxic and dysmorphogenic action of the drug in rodents by administering folic acid supplements were unsuccessful. More work is necessary to elucidate this problem.