Toxicological review of inorganic phosphates.

Inorganic phosphate salts are widely used as food ingredients and in a variety of commercial applications. The United States Food and Drug Administration (FDA) considers inorganic phosphates "Generally Recognized As Safe" (GRAS) (FDA, 1973a, 1979) [FDA: Food and Drug Administration 1973a. GRAS (Generally Recognized as Safe) food ingredients-phosphates. NTIS PB-221-224, FDA, Food and Drug Administration, 1979. Phosphates; Proposed Affirmation of and Deletion From GRAS Status as Direct and Human Food Ingredients. Federal Register 44 (244). 74845-74857, 18 December (1979)] and the European Union (EU) allows inorganic phosphates to be added directly to food (EU Directive 95/2/EC as amended by 98/72/EC). In this review, data on the acute, subchronic and chronic toxicity, genotoxicity, teratogenicity and reproductive toxicity from the published literature and from unpublished studies by the manufacturers are reviewed. Based on the toxicity data and similar chemistry, the inorganic phosphates can be separated into four major classes, consisting of monovalent salts, divalent salts, ammonium salts and aluminum salts. The proposed classification scheme supports the use of toxicity data from one compound to assess the toxicity of another compound in the same class. However, in the case of eye and skin irritation, the proposed classification scheme cannot be used because a wide range of responses exists within each class. Therefore, the eye and skin hazards associated with an individual inorganic phosphate should be assessed on a chemical-by-chemical basis. A large amount of toxicity data exists for all four classes of inorganic phosphates. The large and comprehensive database allows an accurate assessment of the toxicity of each class of inorganic phosphate. Overall, all four classes of inorganic phosphates exhibit low oral, inhalation and dermal toxicities. Based on these data, humans are unlikely to experience adverse effects when the daily phosphorus consumption remains below 70 mg/kg/day (JECFA, 1964, 1982a) [JECFA (Joint FAO/WHO Expert Committee on Food Additives 1964. Specifications for the Identity and Purity of Food Additives and their Toxicological Evaluation) Emulsifiers, Stabilizers, Bleaching, and Maturing Agents. Technical Report Series of the World Health Organization 281; ECFA (Joint FAO/WHO Expert Committee on Food Additives 1982a. Phosphoric Acid and Phosphate Salts. ICS/FA/82)].

Effect of butyl benzyl phthalate on reproduction and zinc metabolism.

Butyl benzyl phthalate (BBP) has been shown to be teratogenic. One mechanism contributing to the teratogenicity of several developmental toxicants, is chemical-induced changes in maternal zinc (Zn) metabolism which result in an increased synthesis of maternal liver metallothionein (Mt), and a subsequent reduction in Zn delivery to the conceptus. We investigated the effects of maternal BBP exposure on maternal-fetal Zn metabolism in Wistar rats. In study I, dams were gavaged with BBP (0,250,1000,1500 or 2000 mg/kg) on gestation days (GD) 11 through 13, and killed on GD 20. Maternal toxicity was evident in the three highest dose groups. Embryo/fetal death and small pup weights and lengths were noted in the 2000 mg BBP/kg group. Fetuses in the 1500 and 2000 mg/kg groups were characterized by poor skeletal ossification, and a high frequency of cleft palate. Rib anomalies were observed in the three highest dose groups. Maternal liver Mt concentrations were only slightly elevated in the 1500 and 2000 mg/kg groups. In study II, dams treated as above, were gavaged with 65Zn and killed 18 h later. While the 2000 mg/kg group had high percentages of 65Zn in some maternal tissues, sequestration of 65Zn in maternal liver was not evident. Thus, BBP is not a strong inducer of Mt, and the teratogenicity of BBP does not appear to be due to alterations in maternal and/or embryonic Zn metabolism.

Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study.

UDP-glucuronosyltransferase (UDP-GT) inducers have been shown to lower plasma levels of thyroxine (T4) by increasing its glucuronidation and elimination by the liver. However, there are no dose-response studies to address the relationship between induction of hepatic UDP-GT and alteration in thyroid homeostasis. Therefore, rats were fed a basal diet or a diet mixed with phenobarbital (PB: 600, 1200, 1800 or 2400 ppm), pregnenolone-16 alpha-carbonitrile (PCN: 250, 500, 1000 or 2000 ppm), 3-methylcholanthrene (3MC: 62.5, 125, 250 or 500 ppm) or a polychlorinated biphenyl mixture (Aroclor 1254, PCB: 10, 30, 100 or 300 ppm) for 15 days to determine their effects on hepatic UDP-GT induction, reduction of serum thyroid hormones and alteration of thyroid function. All the UDP-GT inducers produced a dose-dependent induction of hepatic UDP-GT activity toward T4; the increases produced by PCN (7-fold) and PCB (5-fold) were more pronounced than those produced by PB and 3MC (3-fold). Serum T4 (total and free T4) levels were reduced dramatically by the UDP-GT inducers in a dose-dependent manner (up to 50%-90%). However, they had no effect on serum free T3 and a minimal effect on decreasing serum total T3 levels (10%-20%). Reverse T3 levels were increased by all doses of PCN, by high doses of 3MC and by low doses of PCB. PCN produced a dose-dependent increase in serum thyroid-stimulating hormone (TSH) levels (up to 5-fold), and PB doubled TSH levels. Most surprisingly, even though 3MC and PCB decreased serum T4, they had minimal effects on TSH levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of progesterone pretreatment on cadmium toxicity in male Fischer (F344/NCr) and Wistar (WF/NCr) rats.

A previous report indicated that progesterone pretreatment can markedly reduce cadmium (Cd) toxicity in male NAW mice. Therefore we examined the effects of progesterone pretreatment on Cd toxicity in male Fischer (F344) and Wistar (WF) rats. A single subcutaneous injection of 10 or 30 mumole (CdCl2)/kg proved nonlethal over 24 hr but caused the typical spectrum of testicular lesions in these rats. Moreover, when F344 rats were pretreated with progesterone (100 mg/kg, sc, at -48, -24, and 0 hr) and then given cadmium (20 mumole CdCl2/kg, 0 hr), this dose of cadmium proved very toxic, unexpectedly causing 53% mortality. Progesterone pretreatment had no effect on cadmium-induced lethality in WF rats or on testicular lesions in either strain. Significant elevations in serum lactate dehydrogenase (LDH) activity, indicative of hepatotoxicity, were also observed in progesterone-pretreated F344 rats given cadmium as compared to rats given Cd alone. Progesterone did not induce increases in hepatic or renal metallothionein (MT) and hepatic or testicular MT-I mRNA levels in F344 rats. In contrast, levels of the testicular cadmium-binding protein (TCBP) in progesterone-pretreated F344 rats were doubled. This increase in TCBP provided no protection against cadmium toxicity in the testes. These results indicate that, in contrast to previously reported data for mice, progesterone pretreatment increased the lethality of cadmium in male F344 rats and had no effect on cadmium-induced testicular toxicity in F344 and WF rats.

Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats.

Male Sprague-Dawley rats (250-275 g) were fed diets containing four representative UDP-glucuronosyltransferase (UDP-GT) inducers, phenobarbital (PB), 3-methylcholanthrene (3MC), and pregnenolone-16 alpha-carbonitrile (PCN), as well as a polychlorinated biphenyl (PCB) mixture for 21 days to determine their effect on thyroid hormone levels and thyroid gland function. On Days 3, 7, 14, and 20, blood was collected and serum levels of free and total thyroxine (T4), free and total triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were determined by radioimmunoassay. On Day 21, following treatment with Na131I, the thyroid glands were removed and weighed, and the amount of 131I incorporated was determined. The livers were removed and microsomes were isolated for determination of T4 UDP-GT activity. UDP-GT activity toward T4 was increased by PB, 3MC, PCN, and PCB approximately 190, 290, 260, and 550%, respectively, per kilogram of body weight. PB, 3MC, and PCN reduced serum total and free T4 30-40%, whereas PCB produced a 80-90% reduction. Total T3 levels were slightly reduced by treatment with PB, PCN, and PCB, but none of the treatments decreased free T3 levels. Serum T4 levels (total and free) were found to correlate with UDP-GT activity toward T4. The reductions in thyroid hormone levels led to an increase in TSH levels by PB, 3MC, PCN, and PCB (approximately 50, 50, 210, and 40%, respectively) on Day 20. The elevation of TSH led to an increase in thyroid gland weight by PCN (60%) and PCB (30%) and an increase in thyroidal 131I uptake by PB (60%), PCN (160%), and PCB (100%). Thus, while reasonable correlations between T4 glucuronidation and reduction of serum T4 can be made, only qualified correlations between reduction of T4 and increase in TSH and increase in TSH and stimulation of the thyroid can be made. In conclusion, three classes of microsomal enzyme inducers, represented by PB, 3MC, PCN, as well as PCB, increase UDP-GT activity toward T4 and decrease T4 levels. It appears that induction of UDP-GT plays a role in the effect of these chemicals on the thyroid gland.

Effect of progesterone pretreatment on cadmium toxicity in the male Fischer (F344/NCr) rat.

A previous report has indicated that progesterone pretreatment can markedly reduce cadmium toxicity in male NAW mice. Therefore we examined the effects of progesterone pretreatment on cadmium toxicity in male Fischer (F344/NCr) rats. A single sc injection of 20 mumol CdCl2/kg proved nonlethal over 24 hr but caused the typical spectrum of testicular lesions in these rats. However, when rats were pretreated with progesterone (100 mg/kg, sc, -48, -24, and 0 hr) and then given cadmium (20 mumol CdCl2/kg, 0 hr), this dose of cadmium proved very toxic, unexpectedly causing a 53% mortality. Progesterone pretreatment had no effect on cadmium-induced testicular lesions in surviving rats. Significant elevations in serum lactate dehydrogenase (LDH) activity, indicative of hepatotoxicity, were also observed in progesterone-pretreated rats given cadmium as compared to rats given cadmium alone. Progesterone pretreatment had no effect on the distribution of cadmium to liver, kidney, or testes. Progesterone pretreatment also had no effect on the cadmium-induced increases in hepatic or renal metallothionein (MT) or hepatic or testicular MT mRNA levels. In contrast, levels of the testicular cadmium-binding protein (TCBP) in progesterone-pretreated rats were doubled. These results indicate that, contrary to previously reported data for the mouse, progesterone pretreatment increased the lethality of cadmium in male Fischer (F344/NCr) rats and had no effect on cadmium-induced testicular toxicity. The mechanism by which progesterone enhanced cadmium toxicity, especially cadmium-induced hepatotoxicity, deserves further study.

Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: characterization, induction, and form specificity.

Glucuronidation of thyroxine (T4) by liver microsomal UDP-glucuronosyltransferase (UDP-GT) is a predominant pathway by which T4 is deactivated. This study was conducted to characterize in vitro T4 UDP-GT activity in rat liver microsomal preparations, to determine if T4 glucuronidation is mediated by a particular form of UDP-GT, and to determine if T4 glucuronidation can be increased by microsomal enzyme inducers. Characterization of microsomal T4 UDP-GT activity led to the establishment of optimal assay conditions. UDP-GT activity toward T4 was determined in hepatic microsomal preparations from Wistar and Gunn rats, a mutant strain of Wistar rats deficient in several forms of UDP-GT. Hepatic microsomal preparations from Gunn rats glucuronidated T4 at one-third the rate catalyzed by microsomal preparations from Wistar rats. To determine the effect of four inducers that each increase a separate class of UDP-GT, phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), clofibrate (CLO), saline, or corn oil was administered to male Sprague-Dawley rats ip for 4 days. T4 UDP-GT activity was increased by PB, 3MC, PCN, and CLO 88, 150, 100, and 160%, respectively on a per-milligram-microsomal-protein basis and 138, 125, 100, and 145% on a per-kilogram-body-weight basis, respectively. Therefore, all four classes of UDP-GT inducers increase T4 glucuronidation, suggesting that T4 is not a selective substrate for a particular form of UDP-GT.

UDP-glucuronosyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism.

As many microsomal enzyme inducers have been shown to reduce thyroid hormone levels, this study was conducted to determine if this reduction is produced by directly blocking the synthesis of thyroid hormones, or by indirectly increasing the biotransformation and deactivation of thyroxine (T4) by microsomal enzymes. Surgically thyroidectomized male rats received thyroid hormone replacement therapy by implanted osmotic minipumps, resulting in T4 and T3 serum levels that were similar to those observed in euthyroid controls. Three days after minipump implantation (Day 0), rats were fed diets containing four UDP-glucuronosyltransferase (UDP-GT) inducers: phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), or polychlorinated biphenyls (PCB) for 10 days. PB, 3MC, and PCN reduced total (Days 3-10) and free (Days 7-10) T4 serum concentrations 30-50%, whereas PCB produced a 70-75% reduction in total and free serum T4 (Days 3-10). Treatment with PB, PCN, and PCB decreased levels of total T3 (Days 7-10). UDP-GT activity toward T4 was increased by PB, 3MC, PCN, and PCB 270, 400, 570, and 660%, respectively, and was found to correlate with serum T4 levels (total and free). These results demonstrate that reduction of thyroid hormone levels by microsomal enzyme inducers is produced in part by an extrathyroidal mechanism, quite possibly an increase in T4 glucuronidation.

Toxicological principles of metal carcinogenesis with special emphasis on cadmium.

Metals are an important and emerging class of carcinogens. At least three metals, specifically nickel, chromium, and arsenic, are confirmed human carcinogens, and several more are suspected to have carcinogenic potential in man. Considering that the list of known human carcinogens of any type is very small, it becomes clear that metals make up a substantial portion of the list. Furthermore, many metals are very potent carcinogens in laboratory animals. Despite this, relatively little attention has been given to the topic of metal carcinogenesis. The reasons for this relative lack of attention are not clear but perhaps are fostered by a perception that, because metals are the simplest of molecules, their mechanism of action must also be simple. This could not be farther from the truth and, although no clear mechanisms have emerged in the area of metal carcinogenesis, it has become apparent that they are anything but simple. Metal carcinogens possess several unique characteristics including a remarkable target site specificity. Detection of the mechanism, or mechanisms, of metal carcinogenesis has, however, proven elusive, in part because of a wide diversity of metallic carcinogenic agents and the intricate nature of metal interactions in biologic systems. The following review explores this broad topic, with special emphasis on toxicological principles including dose-response relationships and potential mechanisms, using cadmium as an example.

Cilia and ciliogenesis in endometrial adenocarcinomas. An ultrastructural analysis.

Cilia in neoplastic cells were observed by electron microscopy in specimens from five of six consecutive patients with endometrial adenocarcinoma. Most cilia showed a range of defects, from misalignment and displacement of individual doublets to the absence of up to three peripheral doublets, the pattern varying from 9+2 to 6+2; the central pair of microtubules also was frequently missing. Single peripheral microtubules and displacement of the dynein arms were also observed. The high proportion of cilial defects in neoplastic cells (72%) compared with those in normal endometrium (26%), together with a broader spectrum of cilial abnormalities, suggests that the neoplastic state increases the number and range of cilial lesions.

Monozygotic twins from in vitro fertilization.

A case of identical twins following in vitro fertilization and embryo transfer ( IVF -ET) is described. Two embryos were transferred, but it is apparent that only one implanted and subsequently divided in the early implantation phase to produce identical male twins within a monochorionic , diamniotic placental and membrane configuration. Additional marker studies provide an overall probability of less than 0.001 for dizygosity . There is unlikely to be any relationship between this event and the technique of IVF -ET.

A cytochemical profile of mucus-secreting, ciliated and subcolumnar basal cells of the human cervical mucous membrane.

A range of enzymatic activities in cervical mucus-secreting, ciliated and subcolumnar basal cells were assessed using light and electron microscopic cytochemical techniques. Enzymes detected in all three cell types were those of the tricarboxylic acid cycle, pentose-phosphate and glycolytic pathways, other mitochondrial associated enzymes (NADH and NADPH dehydrogenase), acid phosphatase and non-specific esterase. Mucus-secreting and ciliated cells exhibited thiamine pyrophosphatase and 5' nucleotidase activities while leucine aminopeptidase was most convincingly demonstrated in mucus-secreting cells. Alkaline phosphatase, on the other hand, was detected only in mucus-secreting and subcolumnar basal cells. The profile of enzymatic activities in subcolumnar basal cells closely resembles that of mature lining cells and further supports the hypothesis that these cells differentiate into functioning columnar cells.

An ultrastructural, cytochemical, and autoradiographic study of the mucous membrane of the human cervical canal with reference to subcolumnar basal cells.

This study presents a detailed ultrastructural examination of the human endocervix with evidence for the differentiation of subcolumnar basal cells into mucus-secreting cells and ciliated columnar cells. Cytochemical studies in which peroxidatic activity was used indicate that there are two cell types in the endocervical mucosa which can be distinguished by different localization of reaction product. It may be that estrogen sensitivity accounts for such differences. Autoradiographic investigations demonstrated that glandular cells and subcolumnar basal cells synthesize DNA in premenopausal and postmenopausal women. In addition, subcolumnar basal cells actively synthesized RNA, reflecting their ability for further growth and development.