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Lysinoalanine in the neonatal rat
416
Agricultural
chemicals--Fd
Conner.
Toxico?.
Vol.
17, NO. 4
ing that substantial lossesoccurred during the freeze- gested that faecal nitrosamines could well pose a drying process, but were similqto those found in greater hazard than those in food, due to the long, some foods (ibid 1978, 16, 621). However, it is sug- continuous natuie of the exposure.
AGRICULTURAL
CHEMICALS
for rats. The dominant compound in rat urine wiis ETU and there was one minor metabolite; in mouse Ruddick, J. A., Newsome, W. H. & Iverson, F. (1977). urine, ETU and two major metabolites appeared. A comparison of the distribution, metabolism and Although foetal levels of radioactivity were similar excretion of ethylenethiourea in the pregnant mouse in both species at 3 hr and were also similar to the and rat. Teratology 16, 159. levels in maternal tissues and the placenta, clearance of activity from the foetus was much faster in the Allen, J. R, Van Miller, J. P. & Seymour, J. L. (1978). Absorption, tissue distribution and excretion of 14C mouse than in the rat. By 12 hr, activity expressed ethylenethiourea by the rhesus monkey and rat. Res. as ETU had fallen from- the 3-hr value of 175.6 pg/g to 10*6&g, while the corresponding fall in the rat Commun. them. Path. Pharmac. 20. 109. was from 152.6 to 76.2pg/g. These findings may explain the greater susceptibility of the rat than the Ethylene thiourea (ETU), a decomposition product of ethylenabis(dithiocarbamate) fungicides, has been mouse to the teratogenic effects of ETU. In the second report cited, two female rhesus monshown to be mutagenic in bacteria and teratogenic in rats (Cited in F.C.II: 1977, 15, 361; ibid 1979, 17, keys were given 14C-labelled ETU in an intragastric 177). Its transplacental passage into the foetus has dose of 40 mg/kg and their urine and faeceswere collected over 48 hr. Total urinary excretion of the label been demonstrated (ibid 1977, 15, 80). The first study cited above describes the intragas- over that period was 47 and 6440/ the faecal excretion was Q45 and 0.63% of the dose for the two animals. tric treatment of rats and mice with 240 mg ETU/kg on day 15 of gestation. The compound was labelled Tissue concentrations of radioactivity were fairly low with 35Sfor rats and with 14C for mice. Animals were and were similar in all the organs and tissues anakilled 3, 6, 12, 24 and 48 hr after dosing, and samples lysed. The main contributors to the total body burden of maternal liver, kidney, gastrocnemius muscle and (21 and 28% of the dose at 48 hr) were therefore the blood, the placenta and whole foetuses were exam- muscle, skin and blood. No gross or microscopic ined. Urine and faeces were collected for the periods changes were seen in the tissues. O-6, 6-12, 12-24 and 24-48 hr. In four rats given the same dose of labelled ETU, The concentrations of ETU in mother and foetus urinary excretion averaged 81.9% and faecal excretion 3 hr after dosing were similar in both species. At 6 1.32%. In contrast to the situation in the monkey, and 12 hr, mouse tissues contained about one half less than 1% of the activity remained in the tissues and one sixth, respectively, of the tissue concen- after 48 hr, and the highest level was retained in the trations in the rats, but by 24 hr tissue levels of ETU liver (0013% of dose/g tissue). Other tissues except were again similar in the two species.At 48 hr, radio- the thyroid had an average concentration of 0004o/dg. activity was detectable in all rat tissues but only in In two rats, thyroid concentrations of 0.91 and the liver of the mouse. Excretion of ETU was faster 1.07%/g were recorded. Again, no gross or microin the urine of mice than of rats, but total urinary scopic changes were seen in the tissues. excretion b 48 hr was similar, with totals of 73.5% Although the authors suggest that the inconsistency of the doSt!for mice and 70.5% for rats. Faecal excre- in the levels of radioactivity found in the rat thyroids tion amounts to 2-3x of the dose in both species. was probably due to an analytical problem, accumuThe peak activity in the blood was reached at the lation of ETU in the thyroid gland has been demonsame time (1.3-l-4 hr) in the rat and mouse, but the strated and has been held responsible for the adverse peak concentrations (2146 and 136*7&g, respect- effects of ETU on the rat thyroid. This study provided ively) differed significantly. The calculated half-life of no evidence of any comparable affinity of the monkey ETU in maternal blood was 5.5 hr for mice and 9.4 hr thyroid foi ETU. On the track of ethylene tbiaurea
PROCESSING AND PACKAGING Lysinoalanine in the neonatal rat Struthers, B. J., Hopkins, D. T., Prescher, E. E. & Dahlgren, R. R. (1978). Effects of protein-bound lysinoalanine, N’-DL-(2-amino-2carboxyethyl)+lysine on fetal and neonatal rats. J. Nutr. 108, 954. Lysinoalanine (LAL). which occurs in proteinaceous foodstuffs that have been subjected to alkali treatment, has produced karyomegalic changes and necrosis ih the renal tubular cells of rats (Woodward
CONTAMINANTS
& Short, Fd Cosmet. Toxicol. 1977, 15, 117). In a’ subsequent comparative study of the response of weanling Sprague-Dawley and Wistar rats to a diet containing alkali-treated soya-bean protein with a 1% LAL content (Cited in F.C.T. 1978, 16, 499) marked renal-cell cytdmegaly occurred only in the SpragueDawley rats, although renal calcification was more severein the Wistar strain. The rats used in the reproduction and teratology study cited above were of the Sprague-Dawley strain.
The chemical environment--Fd The female rats were fed throughout gestation and lactation on diets containing a total of 30% protein, of which 530% was an alkali-treated soya-bean protein isolate with a LAL content of I%, the balance being a soya-bean assay protein free of LAL. The LAL levels of the four test diets was therefore 500, 1000, 2000 and 3000 ppm. No skeletal or organ abnormalities were found in the foetuses from the treated dams and there was no cytomegaly in the foetal kidneys. Renal cytomegaly was apparent, however, 30 days after birth in pups born to rats on the 3000-ppm LAL diet. There were
THE Chromium
and
CHEMICAL
the foetus
Gale T. F. (1978). Embryotoxic effects of chromium trioxide in hamsters. Enuir. Res. 16, 101. Studies on the toxicity of chromium and its compounds have resulted in reports of damage to the integument, liver, kidney and respiratory system in both man and experimental animals (Baetjer et al. Chromium; National Academy of Sciences, Washington, DC, 1974). There is also evidence to indicate a risk of broncho-pulmonary carcinoma for individuals handling chromium compounds in industry (Cifed in F.C.T 1976, 14, 215). Despite the fairly extensive work on the toxicology of these materials, studies of their effects on the developing mammalian embryo appear to have been neglected. One contribution on this aspect, however, appeared recently. Hexavalent chromium in the form of the trioxide was administered iv to pregnant golden hamsters in a dose of 5, 75, 10 or 15 mg/kg on day 8 of gestation. The animals were observed for toxic effects until they were killed on day 12, 14 or 15 of gestation. All foetuses were examined for external malformations and a proportion for skeletal and internal abnormalities. Resorption sites in the maternal animals were counted, and stained sections of maternal gall bladder, liver and kidney were examined to detect possible histopathological changes. Of the hamsters given the highest dose, 75% died shortly after treatment. Those given 5 mg/kg showed no ill effects, but the two intermediate doses caused weight loss, changes in urine colour, mottling of the kidneys, distended gall bladders and histological changes in the liver and kidneys. Exposure to the chromium trioxide killed foetuses in utero, the severity of the observed effects being directly related to the dose level. In all cases the resorption sites contained necrotic material unrecognisable as foetal tissue, indicating embryonic deaths early in the gestation period. Abnormal foetuses were retarded or oedematous or displayed specific congenital malformations, in a dose-dependent manner. Only a few external malformations were seen at the lower dose levels but the incidence of cleft palate was significantly higher in all of the experimental groups than the 2% frequency observed in the controls. Of the internal malformations detected, hydrocephalus was the major one and occurred only in
Cosrnc~r.To.vico/. Vol. 17. No. 4
417
no significant differences from the controls in numbers of implantations, corpora lutea or resorption sites, live births per litter, total pups per litter, postnatal deaths or birth weights in the LAL-fed groups. However, pups from dams fed 2000 or 3000 ppm LAL gained weight more slowly than the others. No LAL was detected in the maternal milk, and this failure to gain weight normally was ascribed to a decrease in milk production resulting from a reduction in the availability of the protein in diets containing a high proportion of alkali-treated soya-bean product.
ENVIRONMENT the treated groups. In addition, a few foetuses exposed to chromium exhibited enlargement of the right atrium of the heart and one displayed right renal agenesis. The incidence of supernumary ribs did not show a dose-dependent effect, since the offspring of animals given the lo-mg/kg dose had a lower frequency (7%) of extra ribs than the controls (13%). whilst the experimental groups given the lower doses displayed a frequency at least twice as great as the controls. In all groups, damage to the bones of the cranial vault was minimal, and absence or poor ossification of the arches and bodies of the cervical and caudal regions accounted for most of the vertebralcolumn defects. Poor ossification was also observed in the manubriae and sternebrae. Skeletal damage of the forelimb was restricted to the 7.5- and lo-mg/kg treatments. This study represents a preliminary investigation of the teratogenic effects of chromium. Further work is already in progress on the mechanism involved in chromium trioxide-induced cleft palate. The site of action of chromium with the materno-foetal unitwhether it acts directly within the foetus or indirectly by disruption of a metabolic process in the maternal system or placenta-also requires clarification.
Depigmentation
by 4tert-butylcatechol
Mansur, J. D., Fukuyama, K., Gellin, G. A. & Epstein, W. L. (1978). Effects of 4-tertiary butyl catecho1 on tissue cultured melanocytes. J. invest. Dem. 70, 275. 4-rerr-Butylcatechol (TBC) is used as an antioxidant in several industrial processes, including the manufacture of plastics and polyester resins and the preparation of petroleum products. Topically applied TBC is a depigmenting agent in human and animal skin, although the mechanism of this effect remains unclear (Horio et al. Int. Archs occup. envir. Hlth 1977, 39, 127). In the above-cited study, the depigmenting effect of TBC on the epidermal cells of guinea-pigs was investigated in vitro. Either dimethylsulphoxide (DMSO) or TBC dissolved in DMSO was added to the 5-day-old culture. With concentrations varying from 5 x 10e6 to 3 x 10m5 ml DMSO/ml of medium. the melanocytes did not show any change in shape
Agricultural
chemicals--Fd
Conner.
Toxico?.
Vol.
17, NO. 4
ing that substantial lossesoccurred during the freeze- gested that faecal nitrosamines could well pose a drying process, but were similqto those found in greater hazard than those in food, due to the long, some foods (ibid 1978, 16, 621). However, it is sug- continuous natuie of the exposure.
AGRICULTURAL
CHEMICALS
for rats. The dominant compound in rat urine wiis ETU and there was one minor metabolite; in mouse Ruddick, J. A., Newsome, W. H. & Iverson, F. (1977). urine, ETU and two major metabolites appeared. A comparison of the distribution, metabolism and Although foetal levels of radioactivity were similar excretion of ethylenethiourea in the pregnant mouse in both species at 3 hr and were also similar to the and rat. Teratology 16, 159. levels in maternal tissues and the placenta, clearance of activity from the foetus was much faster in the Allen, J. R, Van Miller, J. P. & Seymour, J. L. (1978). Absorption, tissue distribution and excretion of 14C mouse than in the rat. By 12 hr, activity expressed ethylenethiourea by the rhesus monkey and rat. Res. as ETU had fallen from- the 3-hr value of 175.6 pg/g to 10*6&g, while the corresponding fall in the rat Commun. them. Path. Pharmac. 20. 109. was from 152.6 to 76.2pg/g. These findings may explain the greater susceptibility of the rat than the Ethylene thiourea (ETU), a decomposition product of ethylenabis(dithiocarbamate) fungicides, has been mouse to the teratogenic effects of ETU. In the second report cited, two female rhesus monshown to be mutagenic in bacteria and teratogenic in rats (Cited in F.C.II: 1977, 15, 361; ibid 1979, 17, keys were given 14C-labelled ETU in an intragastric 177). Its transplacental passage into the foetus has dose of 40 mg/kg and their urine and faeceswere collected over 48 hr. Total urinary excretion of the label been demonstrated (ibid 1977, 15, 80). The first study cited above describes the intragas- over that period was 47 and 6440/ the faecal excretion was Q45 and 0.63% of the dose for the two animals. tric treatment of rats and mice with 240 mg ETU/kg on day 15 of gestation. The compound was labelled Tissue concentrations of radioactivity were fairly low with 35Sfor rats and with 14C for mice. Animals were and were similar in all the organs and tissues anakilled 3, 6, 12, 24 and 48 hr after dosing, and samples lysed. The main contributors to the total body burden of maternal liver, kidney, gastrocnemius muscle and (21 and 28% of the dose at 48 hr) were therefore the blood, the placenta and whole foetuses were exam- muscle, skin and blood. No gross or microscopic ined. Urine and faeces were collected for the periods changes were seen in the tissues. O-6, 6-12, 12-24 and 24-48 hr. In four rats given the same dose of labelled ETU, The concentrations of ETU in mother and foetus urinary excretion averaged 81.9% and faecal excretion 3 hr after dosing were similar in both species. At 6 1.32%. In contrast to the situation in the monkey, and 12 hr, mouse tissues contained about one half less than 1% of the activity remained in the tissues and one sixth, respectively, of the tissue concen- after 48 hr, and the highest level was retained in the trations in the rats, but by 24 hr tissue levels of ETU liver (0013% of dose/g tissue). Other tissues except were again similar in the two species.At 48 hr, radio- the thyroid had an average concentration of 0004o/dg. activity was detectable in all rat tissues but only in In two rats, thyroid concentrations of 0.91 and the liver of the mouse. Excretion of ETU was faster 1.07%/g were recorded. Again, no gross or microin the urine of mice than of rats, but total urinary scopic changes were seen in the tissues. excretion b 48 hr was similar, with totals of 73.5% Although the authors suggest that the inconsistency of the doSt!for mice and 70.5% for rats. Faecal excre- in the levels of radioactivity found in the rat thyroids tion amounts to 2-3x of the dose in both species. was probably due to an analytical problem, accumuThe peak activity in the blood was reached at the lation of ETU in the thyroid gland has been demonsame time (1.3-l-4 hr) in the rat and mouse, but the strated and has been held responsible for the adverse peak concentrations (2146 and 136*7&g, respect- effects of ETU on the rat thyroid. This study provided ively) differed significantly. The calculated half-life of no evidence of any comparable affinity of the monkey ETU in maternal blood was 5.5 hr for mice and 9.4 hr thyroid foi ETU. On the track of ethylene tbiaurea
PROCESSING AND PACKAGING Lysinoalanine in the neonatal rat Struthers, B. J., Hopkins, D. T., Prescher, E. E. & Dahlgren, R. R. (1978). Effects of protein-bound lysinoalanine, N’-DL-(2-amino-2carboxyethyl)+lysine on fetal and neonatal rats. J. Nutr. 108, 954. Lysinoalanine (LAL). which occurs in proteinaceous foodstuffs that have been subjected to alkali treatment, has produced karyomegalic changes and necrosis ih the renal tubular cells of rats (Woodward
CONTAMINANTS
& Short, Fd Cosmet. Toxicol. 1977, 15, 117). In a’ subsequent comparative study of the response of weanling Sprague-Dawley and Wistar rats to a diet containing alkali-treated soya-bean protein with a 1% LAL content (Cited in F.C.T. 1978, 16, 499) marked renal-cell cytdmegaly occurred only in the SpragueDawley rats, although renal calcification was more severein the Wistar strain. The rats used in the reproduction and teratology study cited above were of the Sprague-Dawley strain.
The chemical environment--Fd The female rats were fed throughout gestation and lactation on diets containing a total of 30% protein, of which 530% was an alkali-treated soya-bean protein isolate with a LAL content of I%, the balance being a soya-bean assay protein free of LAL. The LAL levels of the four test diets was therefore 500, 1000, 2000 and 3000 ppm. No skeletal or organ abnormalities were found in the foetuses from the treated dams and there was no cytomegaly in the foetal kidneys. Renal cytomegaly was apparent, however, 30 days after birth in pups born to rats on the 3000-ppm LAL diet. There were
THE Chromium
and
CHEMICAL
the foetus
Gale T. F. (1978). Embryotoxic effects of chromium trioxide in hamsters. Enuir. Res. 16, 101. Studies on the toxicity of chromium and its compounds have resulted in reports of damage to the integument, liver, kidney and respiratory system in both man and experimental animals (Baetjer et al. Chromium; National Academy of Sciences, Washington, DC, 1974). There is also evidence to indicate a risk of broncho-pulmonary carcinoma for individuals handling chromium compounds in industry (Cifed in F.C.T 1976, 14, 215). Despite the fairly extensive work on the toxicology of these materials, studies of their effects on the developing mammalian embryo appear to have been neglected. One contribution on this aspect, however, appeared recently. Hexavalent chromium in the form of the trioxide was administered iv to pregnant golden hamsters in a dose of 5, 75, 10 or 15 mg/kg on day 8 of gestation. The animals were observed for toxic effects until they were killed on day 12, 14 or 15 of gestation. All foetuses were examined for external malformations and a proportion for skeletal and internal abnormalities. Resorption sites in the maternal animals were counted, and stained sections of maternal gall bladder, liver and kidney were examined to detect possible histopathological changes. Of the hamsters given the highest dose, 75% died shortly after treatment. Those given 5 mg/kg showed no ill effects, but the two intermediate doses caused weight loss, changes in urine colour, mottling of the kidneys, distended gall bladders and histological changes in the liver and kidneys. Exposure to the chromium trioxide killed foetuses in utero, the severity of the observed effects being directly related to the dose level. In all cases the resorption sites contained necrotic material unrecognisable as foetal tissue, indicating embryonic deaths early in the gestation period. Abnormal foetuses were retarded or oedematous or displayed specific congenital malformations, in a dose-dependent manner. Only a few external malformations were seen at the lower dose levels but the incidence of cleft palate was significantly higher in all of the experimental groups than the 2% frequency observed in the controls. Of the internal malformations detected, hydrocephalus was the major one and occurred only in
Cosrnc~r.To.vico/. Vol. 17. No. 4
417
no significant differences from the controls in numbers of implantations, corpora lutea or resorption sites, live births per litter, total pups per litter, postnatal deaths or birth weights in the LAL-fed groups. However, pups from dams fed 2000 or 3000 ppm LAL gained weight more slowly than the others. No LAL was detected in the maternal milk, and this failure to gain weight normally was ascribed to a decrease in milk production resulting from a reduction in the availability of the protein in diets containing a high proportion of alkali-treated soya-bean product.
ENVIRONMENT the treated groups. In addition, a few foetuses exposed to chromium exhibited enlargement of the right atrium of the heart and one displayed right renal agenesis. The incidence of supernumary ribs did not show a dose-dependent effect, since the offspring of animals given the lo-mg/kg dose had a lower frequency (7%) of extra ribs than the controls (13%). whilst the experimental groups given the lower doses displayed a frequency at least twice as great as the controls. In all groups, damage to the bones of the cranial vault was minimal, and absence or poor ossification of the arches and bodies of the cervical and caudal regions accounted for most of the vertebralcolumn defects. Poor ossification was also observed in the manubriae and sternebrae. Skeletal damage of the forelimb was restricted to the 7.5- and lo-mg/kg treatments. This study represents a preliminary investigation of the teratogenic effects of chromium. Further work is already in progress on the mechanism involved in chromium trioxide-induced cleft palate. The site of action of chromium with the materno-foetal unitwhether it acts directly within the foetus or indirectly by disruption of a metabolic process in the maternal system or placenta-also requires clarification.
Depigmentation
by 4tert-butylcatechol
Mansur, J. D., Fukuyama, K., Gellin, G. A. & Epstein, W. L. (1978). Effects of 4-tertiary butyl catecho1 on tissue cultured melanocytes. J. invest. Dem. 70, 275. 4-rerr-Butylcatechol (TBC) is used as an antioxidant in several industrial processes, including the manufacture of plastics and polyester resins and the preparation of petroleum products. Topically applied TBC is a depigmenting agent in human and animal skin, although the mechanism of this effect remains unclear (Horio et al. Int. Archs occup. envir. Hlth 1977, 39, 127). In the above-cited study, the depigmenting effect of TBC on the epidermal cells of guinea-pigs was investigated in vitro. Either dimethylsulphoxide (DMSO) or TBC dissolved in DMSO was added to the 5-day-old culture. With concentrations varying from 5 x 10e6 to 3 x 10m5 ml DMSO/ml of medium. the melanocytes did not show any change in shape