Metabolism and distribution of [2,3-14C]acrolein in Sprague-Dawley rats. II. Identification of urinary and fecal metabolites.

The metabolites of [2,3-14C]acrolein in the urine and feces of Sprague-Dawley rats were identified after either intravenous administration in saline at 2.5 mg/kg or oral administration by gavage as an aqueous solution as either single or multiple doses at 2.5 mg/kg or as a single dose of 15 mg/kg. Selected urine and feces samples were pooled by sex and collection interval and profiled by combinations of reverse-phase, anion-exchange, cation-exchange, and ion-exclusion high-performance liquid chromatography (HPLC). Feces were also profiled by size-exclusion chromatography. Metabolites were identified by comparison with well-characterized standards by HPLC and by mass spectrometry. The urinary metabolites were identified as oxalic acid, malonic acid, N-acetyl-S-2-carboxy-2-hydroxyethylcysteine, N-acetyl-S-3-hydroxypropylcysteine, N-acetyl-S-2-carboxyethylcysteine, and 3-hydroxypropionic acid. The fecal radioactivity from the oral dose groups was partitioned into methanol-soluble, water-soluble, and insoluble radioactivity, some of which could be liberated by dilute acid hydrolysis. HPLC analysis of these extracts revealed no discrete metabolites. Size-exclusion chromatography indicated a molecular weight range of 2,000 to 20,000 Da for the radioactivity, which was unaffected by hydrolysis at reflux with 6 M acid or base. This radio-activity was thought to be a homopolymer of acrolein, which was apparently formed in the gastrointestinal tract. The pathways of acrolein metabolism were epoxidation followed by conjugation with glutathione, Michael addition of water followed by oxidative degradation, and glutathione addition to the double bond either following or preceding oxidation or reduction of the aldehyde. The glutathione adducts were further metabolized to the mercapturic acids.

Foraging time allocation in relation to sex by the gulf coast fiddler crab (Uca panacea).

Schoener (1971) proposed that the reproductive demands of animals should be important in shaping their foraging behavior because fitness is affected. He defined two forager types: energy maximizers (reproductive success depends on energetic intake) and time minimizers (reproductive success depends on time spent in activities other than foraging), and suggested that females most often illustrate the former and males the latter. We tested whether mating activities influence the foraging behavior of Uca panacea, and the predictions that females would be energy maximizers because of their reproductive strategy and that males would also be energy maximizers because of their courtship activity. Time allocated to foraging by 800 male and female fiddler crabs (at two sites) was quantified; no significant difference in foraging time was found between the sexes. Both male and female crabs allotted a large portion of their time to foraging because both sexes depend on stored energy during their reproductive bouts. Our results show that the particular forager type can be predicted based on reproductive demands, but a forager type can not always be assigned to a particular sex without consideration of all important ecological and physiological factors determining reproductive success.

Metabolism and distribution of [2,3-(14)C]acrolein in laying hens.

The metabolism and distribution of [2,3-(14)C]-acrolein were studied in 10 laying hens orally administered 1.09 mg/kg of body weight/day for 5 days. Eggs, excreta, and expired air were collected. The hens were killed 12-14 h after the last dose and edible tissues collected. The nature of radioactive residues was determined in tissues and eggs. All of the identified metabolites were the result of the incorporation of acrolein-derived radioactivity into normal natural products of intermediary metabolism in the hen except for 1,3-propanediol, which is a known degradation product of glycerol in bacteria.

Metabolism and distribution of [2,3-(14)C]acrolein in lactating goats.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

Gene mutation assay of acrolein in the CHO/HGPRT test system.

The mutagenic potential of acrolein has been studied with a wide range of in vitro and in vivo genetic toxicity assays. The data often have been conflicting, especially with the Ames assay. This study was undertaken to assess the mutagenic potential of acrolein using the CHO/HGPRT assay, both with and without metabolic activation. This assay system was chosen because it provides eukaryotic DNA as the target and is capable of detecting a range of mutational events. Because of its considerable toxicity, acrolein was tested over a very narrow dose range of 0.2-2 nl ml-1 without exogenous activation and 0.5-8 nl ml-1 with rat S-9 activation. Multiple assays were performed under both conditions. The results indicated that while acrolein was clearly very cytotoxic, it did not induce a significant mutagenic response in the presence or absence of metabolic activation.

Reproductive study of acrolein on two generations of rats.

Four groups of 30 male and 30 female rats were intubated with 70 daily doses of acrolein at levels of 0, 1, 3, or 6 mg/kg in a dosing volume of 5 ml/kg. Rats within each dosing group (F0 generation) were then assigned to a 21-day period of cohabitation and dosing for females continued through cohabitation gestation and lactation. Males were euthanized after cohabitation. F1 generation rats were chosen from pups, and a similar pretreatment, cohabitation, gestation, and lactation regimen was accomplished resulting in F2 generation pups. Reproductive parameters, body weights, food consumption, and clinical signs were recorded and necropsies were carried out on all treated animals. Histopathologic exams were accomplished on selected reproductive tissues. In addition, gross lesions, target tissues, stomachs, and lungs were examined. For the most part, reproductive parameters were unaffected by acrolein treatment with the exception of reduced pup weights in the F1 generation pups at the high-dose level (6 mg/kg/day). Gastric lesions were noted consistently in high-dose animals and some mid-dose (3 mg/kg/day) rats. Erosions of glandular mucosa and hyperplasia/hyperkeratosis of the forestomach were the most frequent stomach lesions observed. Effects on body weight gains were noted frequently for the high-dose animals and achieved statistical significance in the mid-dose animals on several occasions. Mortality in all high-dose animals was elevated relative to control animals. Acrolein, therefore, cannot be considered a selective reproductive toxin in the rat, but does produce toxicological effects down to a dosing level of 3 mg/kg/day.

Mutagenic activity of acrolein in S. typhimurium and E. coli.

Acrolein was tested for mutagenic activity in seven strains of Salmonella typhimurium and one strain of Escherichia coli using a preincubation assay procedure. Cytotoxicity was evident at dosing levels above 33 and 67 micrograms acrolein per plate in the absence and presence of S-9 activation, respectively. Evidence of mutagenic activity was seen at non-toxic dosing levels in S. typhimurium strains TA98 and TA100 and E. coli strain WP2 uvrA. Responses in TA98 and E. coli were marginal at best, but a firm positive mutagenic activity was noted in TA100 at 20 micrograms per plate without S-9 activation and at 67 micrograms per plate with S-9 activation. The results of this study demonstrate the mutagenicity of acrolein under highly controlled conditions.

Two-year toxicity and carcinogenicity study of acrolein in rats.

Five-hundred and sixty Sprague-Dawley rats were randomized into one control and three treatment groups (70 of each sex per group). Animals were treated by daily gavage with 0.0, 0.05, 0.5 and 2.5 mg kg-1 acrolein in water (10 ml kg-1). These dosing levels were selected as a result of a 6-week range-finding study. Ten rats of each sex per group were sacrificed at 1 year, and the remainder of the animals were treated for 102 weeks. Daily observations wer made, and various clinical, hematological and urine parameters were measured after 3, 6, 12 and 18 months of treatment and immediately prior to termination. All animals, whether found dead or sacrificed, were subject to necropsy and both absolute and relative organ weights were recorded. An extensive array of tissues were examined microscopically for all test animals. The only effects noted for treated rats that were statistically different from controls were consistent depression of creatinine phosphokinase levels, which was difficult to explain, and consistent increases in early cumulative mortalities in both males and females. There was no significantly increased incidence of microscopic lesions in treated rats, whether neoplastic or non-neoplastic. This study clearly demonstrates the lack of neoplastic response in Sprague-Dawley rats as a result of being treated with acrolein by gavage.

Metabolism and distribution of [2,3-14C]acrolein in Sprague-Dawley rats.

The metabolism and disposition of [2,3-14C]acrolein was studied in Sprague-Dawley rats after oral or intravenous dosing. Four groups of ten rats (five male and five female) were dosed with radiolabeled acrolein intravenously at 2.5 mg kg-1 (Group 2), orally by gavage at 2.5 mg kg-1, either as a single dose (Group 3) or after 14 daily doses of unlabeled acrolein (Group 4), or orally by gavage at 15 mg kg-1 (Group 5). Urine, feces, expired air and organic volatiles were collected for 7 days, after which the animals were sacrificed and tissues collected. All samples were analyzed for total radioactivity. After 7 days, the excretory patterns of male and female rats were almost identical. Urinary excretion was highest in the intravenously dosed animals (66-69%) and lowest in the Group 5 animals (36-40%), whereas the reverse was true for feces (< 2% for i.v. Group 2 animals and 28-30% for the Group 5 animals). Carbon dioxide expiration was comparable (26-31%) across all groups. Tissue concentrations of radioactivity were minimal in all groups (< 1.2%), but concentrations of radioactivity were highest in the intravenous Group 2 animals. The time course of excretion for all groups was similar with the exception of the high-dose animal group, which showed a pronounced delay in excretion during the first 12 h.

Developmental toxicity of acrolein in New Zealand white rabbits.

Pregnant New Zealand white rabbits (20 per group) were treated via stomach tube with 0.0, 0.1, 0.75, or 2.0 mg/kg/day from Days 7 through 19 of presumed gestation and subjected to cesarean sectioning on Day 29. Throughout the period of treatment, clinical observations, feed consumption, and body weights were recorded. At the termination of the study, reproductive and fetal parameters were measured. Three does died during the study, and transient effects on body weight gains and feed consumption were noted, with a subsequent rebound effect reflected in both fetal and maternal weights in the high-dose group (2 mg/kg/day). Resorptions were elevated in the high-dose group, but the effect was not statistically significant. Fetal malformations were distributed evenly among groups, and incidences were consistent with historical control data on the same strain and at the same laboratory. Higher dosage levels (range-finding study, 4.0 and 6.0 mg/kg/day) produced high incidences of maternal mortality, spontaneous abortion, resorptions, clinical signs, gastric ulceration, and/or sloughing of the gastric mucosa. Acrolein was not found to be a developmental toxicant or teratogen at doses not toxic to the does under the conditions employed in this study.

One-year toxicity of orally administered acrolein to the beagle dog.

Forty-eight dogs were separated into four groups of six males and six females. Acrolein (0.1% aqueous) was administered in gelatin capsules to three of these groups at dosing levels of 0.1, 0.5 and 1.5 mg kg-1 day-1 based on results of a range-finding study. After 4 weeks, the high dose was increased to 2 mg kg-1 day-1. The fourth group received deionized water in the same number of gelatin capsules as the high-dose group. Dosing was 7 days per week for 53 weeks. Blood and biochemical measurements were made pretest and at 3-month intervals thereafter. At termination, all dogs were subjected to full necropsy and histological examination. The major test effect noted was frequent vomiting after dosing. This was observed to be dose-dependent and the frequency decreased with time, indicating an adaptive effect. One mid-dose female died during the test and was diagnosed as having died of severe bronchial pneumonia, probably a result of vomitus aspiration. Serum albumin, calcium and total protein values were depressed in high-dose animals throughout the study. Some variability in red blood cell parameters and coagulation times were noted but the significance of these effects was not obvious.