Butylated hydroxytoluene modulates DNA methylation in rats.

The major observation of this investigation is that a single intraperitoneal injection of butylated hydroxytoluene (BHT, 60 mg/kg body mass) results within a few hours in a strong increase in nuclear DNA(cytosine-5)-methyl transferase (methyl transferase) activity in the liver, kidneys, heart, spleen, brain and lungs of male rats. In most organs, the rise in methyl transferase activity is observed as early as 4 h after BHT injection, it reaches a maximum at 8 h and then, except for lungs and brain, gradually decreases to its initial level at 16 h. At the maximum induction times, the methyl transferase activity in liver, kidney and spleen increases by about 16-, 3- and 5-fold, respectively. A second BHT injection at 96 h results in a secondary rise in hepatic methyl transferase activity. Isoelectric focusing electrophoresis of control rat liver nuclear extracts showed methyl transferase activity in the pI 4.7 and 7.4 protein fractions. Both fractions methylate calf thymus DNA better than they do Drosophila melanogaster DNA. In similar extracts from BHT-treated rats, the methyl transferase activity is found in three protein fractions with pI values equal to 4.0, 6.2 and 9.5, respectively. Most of the methyl transferase fractions from the livers of BHT-treated rats methylate the completely unmethylated D. melanogaster DNA better than they do calf thymus DNA. Thus, BHT induces methyl transferase activity that preferably provides de novo DNA methylation. BHT injection had no significant effect on the hepatic contents of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy) and AdoMet/AdoHcy ratios. While BHT injection did not alter the 5-methyldeoxycytidine content in liver DNA, it did appear to alter such content in other organs. BHT appears to cause the reversible changes in the methylation status of an internal cytosine residue in some CCGG sites of the rat liver cytosine DNA-methyl transferase gene. BHT induces also hypomethylation of the renal methyl transferase gene and the hepatic c-Ha-ras gene. While BHT also increases the hepatic mRNA transcripts for the S-adenosylmethionine synthetase and the p53 genes, it had no detectable effects on the corresponding mRNA transcripts for methyl transferase homologous to murine methyl transferase. Thus, BHT induces tissue-specific reversible changes in methyl transferase activity and methylation of total DNA and various genes in rats. A strong increase in methyl transferase activity in rat liver is accompanied with BHT-induced change in the methyl transferase set observed in this organ.

Ethynylestradiol protection against methyl insufficiency in castrated male Wistar/Furth rats fed a methionine-choline-deficient diet.

The interactive effects of dietary methyl insufficiency and the estrogenic compound ethynylestradiol (EE) on the levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were examined in the liver, lungs and pancreas of rats. In addition, such effects on the hepatic content of 5-methyl-deoxycytidine (5-MC) in nuclear DNA were determined. Castrated male Wistar/Furth rats were fed various levels of EE in either: (i) a complete, amino acid-defined diet (diet 1); (ii) the same diet lacking in choline and methionine and supplemented with 0.9% of DL-homocystine (equimolar to methionine) (diet 2); or (iii) diet 2 but only with 0.3% DL-homocystine (diet 2M). Methyl deficiency and EE each independently produced decreased weight gains and increased relative liver weights (liver weight relative to total body weight) compared with control animals. Livers from rats fed diets 2 and 2M without EE had lower levels of SAM and lower SAM:SAH ratios than did the livers from diet 1-fed rats not treated with EE. Hepatic SAM:SAH ratios in diet 1-fed rats were not altered by EE treatment. However, EE treatment increased the hepatic contents of SAM and restored the SAM:SAH levels to normal in rats fed diet 2 or 2M. The levels of SAM + SAH in the livers of rats fed the low homocystine diet (diet 2M) were less than in those fed either diet 1 or diet 2. Thus, the addition of EE at 10 p.p.m. gave protection against reduced levels of SAM, and reduced SAM:SAH ratios in the liver, but had little effect when added to the methyl-adequate diet. No differences in hepatic 5-MC levels were observed in any of the groups as a result of either methyl deficiency or EE treatment. Methyl deprivation alone caused no discernible difference in pancreatic SAM levels but did result in a significant rise in SAH levels and thus in decreased SAM:SAH ratios. EE had no consistent effect on pancreatic SAM, SAH or SAM:SAH ratios in any of the diet groups examined. Similarly, the chronic feeding of diet 2, diet 2M or of EE had no significant effect on the SAM contents of lungs, compared with the corresponding levels in control rats. The protection conferred by EE against SAM insufficiency in the livers of rats fed a methionine- and choline-deficient diet is consistent with the relative insensitivity of female rats to the hepatotoxicity of dietary methyl insufficiency.

Effect of nitrous oxide exposure on maternal and embryonic S-adenosylmethionine levels and ornithine decarboxylase activity.

Nitrous oxide is suspected to be a developmental toxicant in humans. The anesthetic does produce increases in the resorption and malformation frequencies in rodents. The mechanism for the drug's developmental toxicant effects is unknown. Embryonic DNA synthesis is decreased; however, this decrease does not appear to be due to depressed levels of adenine or guanine. In this investigation, we examined the effect of N2O on maternal and embryonic S-adenosylmethionine (AdoMet) levels and ornithine decarboxylase (ODC) activity, and the effect of exogenous methionine (Met) on these parameters was also examined. AdoMet and ODC are involved in polyamine synthesis, and polyamines are involved in regulation of macromolecular synthesis. Pregnant rats were treated with N2O for 24 hours beginning on the morning of day 10 of gestation. There was no effect of N2O on maternal hepatic AdoMet or S-adenosylhomocysteine (AdoHcy) levels; there was also no effect on embryonic AdoMet. Embryonic AdoHcy could not be detected in many of the samples; however, N2O treatment did significantly increase the number of embryonic samples in which AdoHcy was detectable. ODC activity was not affected by either treatment in dams but was increased by N2O in embryos. It is possible that the embryotoxic effect of this anesthetic is mediated by alterations in the AdoMet to AdoHcy ratio or to changes in ODC activity and polyamine synthesis.

Increased levels of S-adenosylmethionine in the livers of rats fed various forms of selenium.

The levels of S-adenosylmethionine (SAM) were determined in the livers of male weanling rats fed six different forms of selenium for five weeks. The following forms of selenium were administrated in the diet in logarithmic doses: sodium selenite, sodium selenate, sodium selenide, selenomethionine, selenocystine, and selenium sulfide. An overall increase in hepatic SAM was observed, and all compounds resulted in at least one observation where SAM was significantly elevated (p < 0.01). No dose-response relationship was found to exist, however. A comparison of the relative toxicity of each of the selenicals was based on a dose of 10 ppm dietary selenium for each chemical form. The elevation of SAM resulting from the subchronic administration of selenium may be one mechanism involved in the well-known chemopreventive effects in experimental models.

Prolonged acetaminophen ingestion by mice fed a methionine-limited diet does not affect iron-induced liver lipid peroxidation or S-adenosylmethionine.

This study determined whether acetaminophen (ACAP)-induced glutathione depletion was associated with liver lipid peroxide formation, or the concentrations of liver S-adenosylmethionine and S-adenosylhomocysteine in mice fed diets with L-methionine below or at the requirement level (0.25 or 0.5%) for 7 wk. Iron dextran (281 mg/kg body wt) or saline was administered for 2 d before measurement of lipid peroxide formation. Chronic dietary ACAP (0.5%) in mice fed 0.25% methionine caused a failure to maintain body weight even though food intake was similar to intake by all other treatment groups. Liver GSH (measured as nonprotein sulfhydryl concentration) and cysteine concentrations were depleted by ACAP and by ACAP plus iron. Liver lipid peroxide formation was increased by iron but was not altered additionally by ACAP ingestion. Liver glutathione peroxidase activity was increased by methionine in controls, whereas glutathione S-transferase activity was increased by ACAP ingestion in mice fed 0.5% methionine compared with controls. Liver S-adenosylmethionine and nuclear 5-methyldeoxycytidine concentrations were not affected by dietary ACAP or methionine. Liver S-adenosylhomocysteine levels were lower in mice fed ACAP and 0.25% methionine compared with mice fed ACAP and 0.5% methionine. In conclusion, chronic ACAP did not increase the susceptibility of mice to liver lipid peroxidation or alter the availability of methyl groups for methylation reactions.

Effects of diet type on incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in BALB/c mice fed AIN-76A diet versus NIH-07 diet.

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and/or nutritional studies, and considered to be nutritionally adequate, were compared with respect to their influence on growth, body weight, lifespan, spontaneous neoplasia, and neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling BALB/c mice were fed either a purified diet (AIN-76A) or a nonpurified, natural ingredient diet (NIH-07), with or without 2-AAF for up to 2 years. Dosages of 2-AAF were administered to males at 0, 20, 40, or 60 ppm in each diet and to females at 0, 100, 125, or 150 ppm. Each group consisted of 96 mice. In most instances, males and females fed purified diet (AIN-fed) gained weight more rapidly, attained higher maximum body weights, and died earlier than their non-purified diet (NIH-fed) counterparts. 2-AAF inhibited weight gain significantly only in AIN-fed females. Thus, females receiving 150 ppm 2-AAF gained little more than their NIH-fed counterparts. At the dosages used in males, 2-AAF did not induce liver neoplasia but the AIN diet was clearly associated with a higher spontaneous frequency of liver neoplasia than the NIH diet. Although 2-AAF induced liver tumors in females fed either diet at all dosages, a higher frequency and earlier appearance of liver tumors among AIN-fed females than their NIH-fed counterparts was apparent mainly at the lowest dosage. 2-AAF induced bladder neoplasia in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in B6C3F1 mice fed AIN-76A diet versus NIH-07 diet.

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and carcinogenic studies, and considered to be nutritionally adequate, were compared with respect to neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling B6C3F1 mice were fed either AIN-76A (a purified diet) or NIH-07 (a natural ingredient diet), with or without 2-AAF, for up to 2 years. Dosages of 2-AAF were administered to males at 0, 40, 60, or 80 ppm in each diet and to females at 0, 150, 200, or 250 ppm. Each group consisted of 96 mice, except the groups of females dosed at 0 and 150 ppm, which consisted of 120 and 72 mice, respectively. The incidence of malignant liver tumors was significantly greater in all AIN-fed groups compared to corresponding NIH-fed groups, as was the total incidence of tumors (malignant + benign). Similarly, the incidence of malignant and total bladder tumors was greater in AIN-fed groups of mice administered the two high doses of 2-AAF for each sex compared to NIH-fed groups. No malignant bladder tumors were observed among any of the groups of mice receiving control diets. This was also true for the males on low dosages (40 ppm) of 2-AAF. The AIN-fed group of low dose females (150 ppm) developed 4% incidence of malignant bladder tumors. This study dramatically showed the importance of diet selection in chronic carcinogenic studies and suggests that results obtained with the use of a purified diet may differ both qualitatively and quantitatively from results obtained with a natural ingredient diet.

Hydroxylation and deglycosylation of 2'-deoxyguanosine in the presence of magnesium and nickel.

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2'-deoxyguanosine (dG) that has been caused by ascorbic acid and H2O2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H2O2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H2O2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2-7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2'-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.

The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital.

The ability of the dietary methyl donors methionine and choline to inhibit the carcinogenic and tumor-promoting effects of phenobarbital (PB) in the livers of male weanling C3H mice was examined. The mice were fed a commercial rodent diet with or without 0.05% PB. Thirty animals from each set received the diet with either: (1) no dietary supplementation, (2) an additional 1.0% choline chloride, (3) 1.5% DL-methionine or (4) both 1.5% DL-methionine and 1.0% choline chloride. Additional groups of 30 animals with the same eight dietary and PB-treatment regimens described above were given a single initiating dose of 150 mg diethylnitrosamine (DENA)/kg body wt dissolved in saline, or the saline solution only, 1 week prior to the start of PB feeding. The 16 treatment groups were fed their respective diets for 12 months. Statistical trend analysis showed that increasing levels of supplemental methyl donors gave highly significant protection in PB-treated mice (P less than 0.01). The incidence of liver carcinomas in the four dietary groups not receiving PB or DENA varied from 0 to 7%. The PB-treated animals not receiving an initiating dose of DENA developed hepatocellular carcinomas (HCCs) at incidences of 79% in group 1 animals, 74% in group 2 animals, 60% in group 3 animals, and 31% in group 2 animals respectively. Thus, incidence of HCCs in group 4 was significantly lower than in groups 1, 2 or 3 (P less than 0.01). However, the total incidence of liver tumors (adenomas plus carcinomas) was about the same in all DENA or PB-treated groups. Thus, dietary supplementation with methyl donors increased the proportion of animals bearing liver adenomas as their most advanced hepatic lesion in PB-treated mice. In DENA-treated mice fed PB, dietary supplementation with methionine and choline protected against the formation of liver carcinomas (P less than 0.02); however, methionine and choline had no significant effect on liver tumor formation in mice fed the PB-free diets. Methionine and choline supplementation gave significant protection against HCC metastases in the lungs of the tumor-bearing mice in groups initiated with DENA followed by PB promotion. These results support the hypothesis that PB exerts it tumorigenic activity in mice at least in part through a physiological insufficiency of labile methyl groups.

Influence of a purified diet and route of administration on the metabolism and disposition of estradiol in B6C3F1 mice.

The metabolism and disposition of [6,7-3H]estradiol ([3H]E2) given by gavage (po) or intravenously (iv) were examined in female B6C3F1 mice fed either the purified AIN-76A (AIN) or cereal-based NIH-07 (NIH) diet for a period of 8 weeks prior to treatment with E2. Initially, 40.6 Ci of [3H]E2 was given iv to each mouse. Subsequently, 45.6 Ci of [3H]E2 was given po to the same mice. Samples of blood, urine, and feces were obtained during a 48-hr period after each dosing. Total radioactivity was determined for each sample. Urine and plasma samples were analyzed by HPLC, and the radiolabeled metabolites were tentatively identified and quantified. Statistical comparisons were made of the effects of diet, route of administration, and interactions among groups. Analysis revealed: 1) greater fecal than urinary excretion of radioactivity regardless of route of administration or diet fed, 2) more radioactivity excreted in the urine of AIN-fed than in NIH-fed mice, significantly different only after iv administration (p less than 0.02), and 3) a greater feces:urine ratio of excreted radioactivity following iv than po administration and from NIH-than AIN-fed mice. Metabolite profiles showed: 1) no differences in urine due to route of administration, 2) estriol conjugates dominated urinary metabolites, 3) accumulation of radioactive material in plasma that apparently was tritiated water, with more rapid accumulation of tritiated water after po than iv administration, 4) relatively more plasma estradiol-17-glucuronide, estriol-3-sulfate, and estriol in po- than in iv-treated mice, 5) estradiol-3, 17-sulfate in plasma of iv- but not po-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of oral administration of sulfamethazine on thyroid hormone levels in Fischer 344 rats.

Fischer 344 rats (810 of each sex) were divided into treatment groups and fed diets containing 0, 10, 40, 600, 1200, or 2400 ppm sulfamethazine. Serum samples were analyzed for levels of thyroid-stimulating hormone (TSH), total thyroxine (T4), total triiodothyronine (T3), and T3 uptake after 12, 18, or 24 mo of continuous dosing. There were no statistically significant differences in T3 levels or percent T3 uptake for either sex after any of the exposure periods. The serum T4 levels were lower (p less than 0.05) for females dosed at 1200 and 2400 ppm for 18 mo and for males dosed at 600, 1200, or 2400 ppm sulfamethazine for 24 mo than for those dosed at levels of 40 ppm or less. Serum TSH levels showed a general increasing trend (but not statistically significant) among animals receiving 600 ppm or more sulfamethazine. There was a significant dose-related reduction in (T3 + T4)/TSH ratio for both sexes (p less than 0.05) after 18 and 24 mo of exposure at dose levels of 600 ppm or more. A lack of response at 12 mo may have been due to the shorter treatment time. At each sacrifice period both sexes of rats fed sulfamethazine at 1200 and 2400 ppm had significantly heavier (p less than 0.05) thyroid weights than animals fed control diet. The heavier thyroid weights in the dosed animals may have resulted from increased TSH levels. The cause of reduction in serum T4 was not clearly evident. Therefore, the thyroid hormone to pituitary feedback mechanism apparently compensated for sulfamethazine effects in most animals. This would suggest that the thyroid gland was not irreversibly affected.

Effects of purified (AIN-76A) and natural-ingredient (NIH-07) diets on responses of BALB/c and B6C3F1 female mice to estradiol.

Female BALB/c and B6C3F1 mice were examined after a 3-wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified (AIN-76A) or a natural-ingredient (NIH-07) diet. The use of AIN-76A was associated with a 9-13% greater (p less than 0.001) body weight and a 36-43% higher (p less than 0.001) serum cholesterol in both mouse genotypes when compared to mice fed NIH-07. Conversely, when fed NIH-07, both mouse genotypes had a 20-22% higher (p less than 0.003) serum urea nitogren and 2-3.5% higher erythrocyte count (p less than 0.001) and hemoglobin concentration (p less than 0.04) than when fed AIN-76A. Reduced erythrocyte parameters suggest that chronic feeding of the purified diet might result in anemia. No significant compound or diet-related differences were noted for serum creatinine, alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transferase. Although there was no diet effect on absolute or differential white blood cell count, estradiol caused a decrease in the total white blood cell count (p less than 0.014) and an increase in the percentage of polymorphonuclear leukocytes (p less than 0.014) in BALB/c and decreased the percentage of lymphocytes (p less than 0.005) in B6C3F1 females. In addition, estradiol increased uterine weight and inhibited thymic and splenic weights in one or both genotypes. Spleen and thymus weight responses to estradiol were not significantly influenced by diet. However, the uterine weight responses to estradiol were apparently influenced by diet in both genotypes. In B6C3F1 mice, the uterus weighed more at each level of estradiol when mice were fed AIN-76A compared to NIH-07 diet. In BALB/c mice, this was true only at the two lower dietary concentrations of estradiol. In conclusion, mice fed the purified diet, AIN-76A, differed from those fed the cereal-based diet, NIH-07, in hematology, clinical chemistry, and uterine weight response to estradiol.

Sex specificity of myocardial damage in mice fed a purified diet.

Both sexes of BALB/c and B6C3F1 mice were divided into test groups and fed either a purified diet (AIN-76A) or a natural ingredient diet (NIH-07) containing graded levels of 2-acetylaminofluorine (2-AAF) for 90 days. A large number of dead or moribund B6C3F1 males fed the AIN diet were removed from the study prematurely. AIN-fed B6C3F1 mice removed early as well as some sacrificed at the end of the study showed myocardial damage with hemorrhage. A much smaller number of BALB/c males fed the AIN diet also exhibited these signs while none of the females from either stock were affected. Mice having these lesions were confined largely to 2 of 5 treatment groups. Increased levels of serum aspartate aminotransferase (GOT) (P less than .01) occurred in the AIN-fed B6C3F1 male mice that were sacrificed, supporting the histopathological observation of myocardial damage. There was no other significant difference in the GOT between diets or 2-AAF doses. No environmental factors could be associated with the problem and microbiological and chemical analyses of the diets showed no convincing evidence of specific pathogenic organisms or nutritional deficiencies that might have caused these lesions. Extended storage (up to 4 months) and one batch of feed in particular seemed to be associated with mice having myocardial damage. These associations were highly strain and sex dependent and suggest that great care must be taken in the manufacture and handling of the diet. Furthermore, it seems likely that the diet may be marginally adequate for some strains of mice and may require modification before it will become generally useful.

Comparison of histological responses of BALB/c and B6C3F1 female mice to estradiol when fed purified or natural-ingredient diets.

Female BALB/c and B6C3F1 mice were examined after a 3-wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified diet (AIN-76A) or a natural-ingredient diet (NIH-07). Histological findings, which became more prevalent with increasing estradiol dosage in both mouse genotypes, included vaginal hyperkeratosis and mucoid stroma, uterine inflammation, hydrometra and glandular hyperplasia, and ovarian corpora lutea depletion. At the two lower doses of estradiol, responses were generally more prevalent or severe in mice fed the purified diet than in those fed the natural-ingredient diet. However, in BALB/c mice, several responses to the two higher estradiol doses were greater when estradiol was given in the natural-ingredient diet rather than in the purified diet. These responses included corpora lutea depletion, vaginal hyperkeratosis, and uterine inflammation and hydrometra. In B6C3F1 mice, most responses to estradiol at concentrations of 400, 800, and 1600 ppm were more prevalent or severe in mice fed the purified diet than in those fed the natural-ingredient diet. It can be concluded that several responses to estradiol in mice maintained for a 3-wk period on a purified diet differed significantly from mice maintained on a natural-ingredient diet.

Effects of storage conditions on nutritional qualities of semipurified (AIN-76) and natural ingredient (NIH-07)diets.

The effects of storage condition on the shelf life of the AIN-76 diet were investigated. Samples of the diet were stored at -70 degrees, 4 degrees, 20 degrees, and 23-30 degrees under atmospheric air and at 4 degrees and 20 degrees under argon. Levels of vitamin A, thiamine, rancidity, bacteria and mold were monitored during 168 days of storage. A 41.3% loss of vitamin A occurred in samples stored at 23-30 degrees. A marked decline in thiamine was observed in all samples stored at 20 degrees or above. Loss of thiamine was significantly greater in samples stored at 20 degrees than in those stored at 4 degrees, and greater in samples stored under air than those stored under argon. Rancidity reached a level previously shown to be associated with a disagreeable odor and taste (peroxide value greater than 140) with in 30, 50 and 90 days when the diet was stored at 23-30 degrees or 20 degrees under air and 20 degrees under argon, respectively. Peroxide values remained well below 140 in samples stored at 4 degrees or colder. None of the nutritional parameters tested reached unacceptable levels in any of the samples stored at 4 degrees or colder during the 168 days. These results indicate that to achieve maximum shelf life, the AIN-76 diet should be stored at 4 degrees or colder. Although less effective than low temperature, an argon atmosphere extended the shelf life and was additive with lower temperatures.

Evaluation of a new rat feeder for use in chemical toxicology and nutrition studies.

A rat feeder was designed, fabricated, and evaluated which eliminated many of the problems associated with commercially available rat feeders (such as inadequate feed capacity, excessive feed spillage, excessive feed contamination, and difficulties with cleaning feeders). The feeder was capable of holding up to 700 g of meal-type feed. It provided rats with a continuous supply of diet which was virtually free from feces and urine contamination. Feed waste due to spillage and inaccessible feed was minimal and estimated to average 0.8%. Growth and weight maintenance in weanling and adult rats using the feeder were similar when compared to rats receiving pelleted feed ad libitum.

A multi-generation toxicity evaluation of P,P'-DDT and dieldrin with Japanese quail. II. Tissue residues analyses.

Selected tissues and egg yolks of Japanese Quail fed diets containing DDT or dieldrin in a four-generation (parental, F1, F2 and F3) study were analyzed for residues of parent compound and/or specific metabolites. Diets containing DDT (5 and 50 ppm) or dieldrin (0.1 and 1.0 ppm) were fed to each generation for 10 wks followed by a 12-wk recovery period in order to determine generation-accumulative effects, maximum residue levels and decline of residues during recovery. Tissue-residue storage and intergeneration transmission for DDT and dieldrin were generally similar and were related to dietary levels. Tissue residues in newly hatched birds were elevated initially for each generation subsequent to the parents, but any cumulative effects between generations were transitory, as residues after 5-to-10 wks were similar in each generation. Tissue residues in all groups showed a decline during the 12-wk recovery period. Decline during recovery was greater for females than males, probably attributable to egg production. Egg yolk residues for birds fed 5.0-ppm DDT declined to near control levels during the recovery period; at 50 ppm, residues declined but remained elevated above controls. For the quail fed dieldrin, residue levels were above those in controls; as with DDT the subsequent decline was more marked in the females.