[Show abstract][Hide abstract] ABSTRACT: Madder color (MC) extracted from the roots of Rubia tinctorum (madder root) has been used as a food coloring in Japan. Our previous studies revealed MC to have obvious subchronic and chronic toxicity and potent carcinogenicity targeting rat liver and kidney. In the present two-year carcinogenicity study, conducted to further elucidate the long-term effects of MC and its target organs, male and female F344 rats were fed diet containing 0%, 2.5%, and 5.0% MC for 104 weeks. Body weights were significantly decreased in treated groups of both sexes throughout the feeding period. However, survival rates at week 104 were higher in treated groups of both sexes than in controls. Relative weights of the kidneys and liver were significantly increased in treated groups of both sexes. Histopathologically, karyomegaly and atypical tubules/hyperplasias, as well as renal cell adenomas and carcinomas were significantly increased in treated groups of both sexes with dose-dependence. Moreover, the incidence of hepatocellular adenomas and/or carcinomas was increased significantly with a dose-relation in treated groups of both sexes. These data provide clear evidence that MC exerts unequivocal carcinogenicity against renal tubule cells and hepatocytes in rats.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 12/2008; 47(1):184-91. DOI:10.1016/j.fct.2008.10.031 · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate chronic toxicity of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., F344 rats were fed diet containing 0%, 0.2%, 1.0% or 5.0% MC for 53 weeks. Hematological changes including anemia and serum biochemical alterations indicating hepatotoxicity were demonstrated at 5.0% in both sexes. Relative weights of the liver were significantly increased from 1.0% in both sexes, and those of the kidney were significantly increased from 1.0% in males and from 0.2% in females. Histopathologically, atypical renal tubule hyperplasias were increased at 1.0% or higher in both sexes in association with increase of cell proliferative activity in the tubules. A renal cell adenoma was observed in a male rat receiving 5.0% MC. In addition, glutathione S-transferase placental form-positive liver cell foci were significantly increased at 5.0% in both sexes. These results indicate that MC has chronic toxicity targeting kidney, liver and blood cells. Moreover, the results strongly suggest that MC may have the carcinogenic potential in the kidney and the liver.
Food and Chemical Toxicology 09/2008; 46(10):3303-10. DOI:10.1016/j.fct.2008.07.025 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 13-week repeated oral dose toxicity study of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., was performed using F344 rats. Five groups of animals, each consisting of 10 males and 10 females, were fed diet containing 0, 0.6, 1.2, 2.5 or 5.0% MC for 13 weeks. During the experiment, lower body weight was evident from the 2.5% dose. Hematologically, fluctuation in red blood cell (RBC) parameters suggestive of weak anemia (females), and slight increases of platelet counts (both sexes) and white blood cell (WBC) counts (males) were observed at higher doses. Serum biochemically, slight fluctuations were observed in many parameters, including increased total protein (TP), conjugated bilirubin, Ca, and inorganic phosphate, and decrease of the albumin/globulin (A/G) ratio in both sexes, with dose-dependence for TP and A/G from 0.6% in females. Histopathological changes were mainly observed in the renal proximal tubules, such as microvesicular vacuolar degeneration in the cortex and karyomegaly in the outer medulla involving both sexes, lesions being evident even with 0.6%. In the outer medulla, elevation of cell proliferation activity as assessed with proliferating cell nuclear antigen was observed in males from 2.5%. Severity of focal necrosis of hepatocytes was increased only in females at 5.0%, while the increased relative liver weight as with the increased conjugated bilirubin was evident in both sexes from 1.2%. The results thus suggest that MC exerts mild toxicity, targeting liver, kidneys, and possibly RBCs and WBCs, some renal changes being evident from 0.6% in diet, that is attributable to be the lowest-observed adverse effect level (305.8-309.2mg/kg body weight/day).
Food and Chemical Toxicology 02/2008; 46(1):241-52. DOI:10.1016/j.fct.2007.08.002 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously examined the impact of perinatal exposure to ethinylestradiol (EE), methoxychlor (MXC), diisononyl phthalate (DINP), and genistein (GEN) in maternal diet on rat offspring, and found developmental and/or reproductive toxicity with 0.5 ppm EE, 1200 ppm MXC, and 20,000 ppm DINP. Although the toxicological profile with MXC was similar to the EE case, the population changes in pituitary hormone-producing cells totally differed between the two cases, changes being evident from 240 ppm with MXC. In the present study, to assess the impact of these agents on brain sexual differentiation, region-specific mRNA expression of estrogen receptors (ER) alpha and beta, the progesterone receptor (PR), gonadotrophin-releasing hormone, steroid receptor coactivators (SRC)-1 and -2, and calbindin-D in microdissected hypothalamic medial preoptic areas (MPOAs) at postnatal day 10 was first analyzed in rats exposed to 0.5 ppm-EE from gestational day 15 by real-time RT-PCR. Sexually dimorphic expression of ER alpha and PR was noted with predominance in females and males, respectively, EE up-regulating SRC-1 in males and ER beta and PR in females. Next, we similarly examined expression changes of ER alpha and beta, PR, and SRC-1 in animals exposed to MXC at 24, 240, and 1200 ppm, DINP at 4000 and 20,000 ppm, and GEN at 1000 ppm. MXC at 1200 ppm down- and up-regulated PR in males and females, respectively, and DINP at 20,000 ppm down-regulated PR in females, while GEN did not exert any clear effects. The results thus suggest that agents causing developmental and/or reproductive abnormalities in later life may affect hypothalamic PR expression during the exposure period in early life.
[Show abstract][Hide abstract] ABSTRACT: The efficacies of N-acetylcysteine (NAC), phenylethyl isothiocyanate (PEITC), and 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ) at preventing the neurotoxicity and testicular toxicity of acrylamide (ACR) were investigated in rats. To this end, Sprague-Dawley males were given 0.02% ACR in drinking water, with or without 1% NAC, 0.5% PEITC or 0.1% HTHQ in the diet for four weeks. A group of untreated controls was also included in the study. All ACR-treated animals exhibited progressive neurotoxicity as judged by gait scores, and among the chemicals co-administered, only HTHQ caused any suppression by the end of the experiment, and this was slight. The severity of the neurotoxicity, as judged by axonal degeneration in the spinal gracile fasciculus and sciatic nerve (distal portion) and aberrant dot-like synaptophysin immunoreactivity, reflecting nerve terminal degeneration in the cerebellar molecular layer, was not clearly reduced by co-administration of HTHQ, NAC or PEITC either. ACR-induced sciatic nerve axon atrophy was marginally and non-significantly reduced by HTHQ. In contrast, in terms of ACR-induced testicular toxicity, exfoliation of spermatids into seminiferous lumen was clearly reduced by co-administered PEITC and was marginally reduced by co-administered HTHQ. These antioxidative agents may therefore reduce/prevent ACR-induced toxicity, at least in the testes.
Archive für Toxikologie 10/2005; 79(9):531-41. DOI:10.1007/s00204-005-0656-6 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.
[Show abstract][Hide abstract] ABSTRACT: Initiation activity of phenylethyl isothiocyanate (PEITC) was examined in a two-stage urinary bladder carcinogenesis model. Male 6-week-old Fischer 344 rats were fed diet containing 0.1% PEITC for 12 or 24 weeks, with or without subsequent administration of 5% sodium l-ascorbate (Na-AsA) in diet until week 48, or for the entire experimental period. After 12 weeks of PEITC-treatment, both simple hyperplasia and papillary or nodular (PN) hyperplasia had developed in all animals, but the majority of these lesions had disappeared at week 48, irrespective of the Na-AsA-treatment. The same lesions after 24 weeks of PEITC-treatment had progressed to dysplasia and carcinoma, in a small number of cases by week 48 (6% in incidence for each lesion), but enhancement by the Na-AsA-treatment was evident only with simple hyperplasias (from 56 to 100% in incidence) and PN hyperplasias (from 19 to 56%). The results suggest a limited initiation activity of PEITC with induction of irreversible lesions by 24 weeks of exposure.
Cancer Letters 04/2005; 219(2):147-53. DOI:10.1016/j.canlet.2004.06.024 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate developmental toxicity of di-n-butyl phthalate (DBP) with exposure during the period from late gestation to following lactation, maternal rats were given DBP at dietary concentrations of 0, 20, 200, 2000 and 10,000 ppm from gestational day 15 to postnatal day (PND) 21. At 10,000 ppm, male offspring showed a decreased neonatal anogenital distance and retention of nipples (PND 14), while females showed a slight non-significant delay in the onset of puberty. At PND 21, reduction of testicular spermatocyte development was evident from 20 ppm, as well as mammary gland changes at low incidence in both sexes. At this time point, population changes of pituitary hormone-immunoreactive cells were observed at 10,000 ppm with a similar pattern of increase in the percentages of luteinizing hormone (LH)-positive and decrease in follicle-stimulating hormone (FSH) and prolactin producing cells in both sexes, effects also being evident on FSH from 200 ppm and LH from 2000 ppm in females. During postnatal week (PNW) 8-11, marginal increase of the number of cases with extended diestrus was found at 10,000 ppm. At adult stage necropsy, testicular lesions appeared to be very faint in most cases, but degeneration and atrophy of mammary gland alveoli were observed in males from 20 ppm. Although without clear monotonic dose-dependence, relative pituitary weights were increased with the intermediate doses in males at PNW 11. In females, relative pituitary weights were decreased after 10,000 ppm at PNW 11, and from 200 ppm at PNW 20. The proportion of FSH-positive cells in the pituitaries at PNW 11 was increased in both sexes at 10,000 ppm. Thus, developmental exposure to DBP affected female sexual development involving pituitary function, while in males testicular toxicity was mostly reversible but mammary gland toxicity was persistent at a dose level as low as 20 ppm.
[Show abstract][Hide abstract] ABSTRACT: To identify genes showing responses to estrogen exposure in the livers of animals in a repeated oral dose toxicity study, dose-dependent gene expression profiles were analyzed using high-density oligonucleotide microarrays in Sprague-Dawley rats of both sexes administered ethinylestradiol (EE) for 28 days at concentrations of 0, 0.01, 0.1, and 1.0 ppm in the diet. Among 3776 genes examined, examples showing increased expression on EE-treatment were detected predominantly in females. Genes showing dose-dependent up-regulation with greater than five-fold change at 1.0 ppm from the control levels were found to, respectively, number 4 in males, and 24 in females. Most of the latter exhibited relatively high basal expression as well as low variability, and many exhibited clear dose-dependence. Genes showing dose-dependent down-regulation were rather few, and many of those affected exhibited relatively low expression levels with large variation between animals, like genes showing dose-unrelated expression patterns in both sexes or dose-dependent up-regulation in males. Considering that detection of changes in endocrine-linked organs and estrous cyclicity is only possible at the high dose of 1.0 ppm, up-regulation of genes dose-dependently in females provides a sensitive tool to detect estrogenic effects in the rat liver in the framework of the 28-day toxicity study.
[Show abstract][Hide abstract] ABSTRACT: We previously found that effects of perinatal dietary exposure to ethinylestradiol (EE) on the rat reproductive system differ depending on the diet used, with a more pronounced estrogenic impact with a regular diet that includes soy-derived proteins than with a soy-free (SF) diet. The present study was performed to examine whether genistein (GEN), a soy-derived major phytoestrogen, acts synergistically with EE. Maternal rats were fed SF diet without chemical (control) or containing 0.5-ppm EE, 0.5-ppm EE + 100-ppm GEN, 0.5-ppm EE + 1250-ppm GEN, or 1250-ppm GEN, from gestational day 15 to postnatal day (PND) 11. EE reduced serum testosterone in males at PND 3, and affected the onset of puberty of both sexes and estrous cyclicity and reproductive system in females, irrespective of co-administration of GEN. GEN alone also affected estrous cyclicity and the reproductive system in females. However, no combination effects of GEN with EE were evident, suggesting no synergism between the two.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.
Journal of Histochemistry and Cytochemistry 07/2004; 52(7):903-13. DOI:10.1369/jhc.3A6215.2004 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously performed dose-response studies of genistein, diisononyl phthalate, 4-nonylphenol, methoxychlor (MXC), and bisphenol A to examine the impact of maternal dietary exposure from gestational day 15 to postnatal day 10 on the development of rat reproductive system in later life. Among the chemicals MXC alone showed typical estrogenic effects only at the maternally toxic 1200 ppm. The present study was performed to examine the sensitivity of immunohistochemical analysis of pituitary cells of offspring similarly exposed to each chemical for detection of endocrine-disrupting effects. For this purpose, ratios of pituitary cells expressing luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL), were measured at 3 and 11 weeks of age. Ethinylestradiol (EE) at 0.5 ppm was used as a reference chemical. At week 3, decrease in the relative proportions of LH, FSH, and PRL cells in males and LH cells in females was evident with MXC at 1200 ppm. At week 11, increase was found for PRL cells from 240 ppm MXC, and FSH cells at 1200 ppm in females. On the other hand, EE increased the PRL cell percentage in females at week 3 but no effects were apparent at week 11. The other chemicals were without influence at either time point. The results suggest that the assessment of the pituitary cell populations might be a more sensitive approach to detect perinatal endocrine-disrupting effects than other methods. The difference in the pituitary effect between MXC and EE is discussed.
Archive für Toxikologie 05/2004; 78(4):232-40. DOI:10.1007/s00204-003-0528-x · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A subchronic toxicity study of N-acetylglucosamine (GlcNAc), a monomeric form of chitin, was conducted in groups of 10 male and 10 female F344 rats fed pelleted diets containing 0, 0.625, 1.25, 2.5 or 5% concentrations for 13 weeks. All animals survived until the end of the experiment. Slight, non-significant increase in body weights was observed in males receiving 0.625, 1.25 or 2.5% from week 4 until the end of the experiment, when significant elevation was found for the males receiving 0.625, 1.25 or 2.5% at the terminal sacrifice to result in decreased relative weights of many organs in these cases. However, there were no obvious indications of toxicity in any group receiving GlcNAc in terms of clinical signs, food intake, hematology, serum biochemistry, and histopathological findings. Thus, it was concluded that orally administered GlcNAc exerts no obvious toxicity to F344 rats at concentrations up to 5% in the diet for 13 weeks. Based on the present toxicity data, > or =5% was determined to be a no-observed-adverse-effect level, translated into 2476 and 2834 mg/kg/day for male and female rats, respectively.
Food and Chemical Toxicology 04/2004; 42(4):687-95. DOI:10.1016/j.fct.2003.12.005 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two potential endocrine-disrupting chemicals, bisphenol A (BPA) and nonylphenol (NP), were assessed for their long-lasting effects on endocrine/reproductive systems following transplacental and lactational exposure to rat offspring during a time-window that included the critical period for brain sexual differentiation. Each chemical was mixed with diet at concentrations of 60, 600 and 3000 ppm and was provided to maternal Sprague-Dawley rats from gestational day (GD) 15 to postnatal day (PND) 10. Ethinylestradiol (EE) at 0.5 ppm was used as an estrogenic reference drug. During pregnancy and lactation, including the exposure period, a soy-free rodent diet was provided to eliminate possible modification of the study results by plant-derived phytoestrogens. Effects on endocrine/reproductive systems were evaluated by examining the anogenital distance, organ weights before puberty, onset of puberty, estrous cyclicity, and organ weights and histopathology of adult endocrine organs (at 11 weeks of age), as well as the volume of the sexually dimorphic nucleus of preoptic area. Both NP and BPA, at high doses, caused decreases in maternal body weights and retardation of offspring growth, but neither affected any of the endocrine/reproductive endpoints of offspring, whereas EE induced irreversible changes in estrous cyclicity and histopathology of ovaries and uterus of adult females. The results indicated that maternal dietary exposure to NP or BPA at concentrations up to 3000 ppm from GD 15 through PND 10 do not exert any apparent adverse effects on the endocrine/reproductive systems of offspring.
Archive für Toxikologie 03/2004; 78(2):97-105. DOI:10.1007/s00204-003-0517-0 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effects of two diets, differing in phytoestrogen contents, on the phenotypic changes induced in the endocrine/reproductive system by perinatal exposure to an estrogen agonist during a critical period for brain sexual differentiation in rats. Ethinylestradiol (EE) was mixed at a concentration of 0.5 ppm into two diets: CRF-1, a standard rodent diet containing soybean-derived phytoestrogens; and a soy-free (SF) diet. These diets were provided to maternal Sprague-Dawley rats during gestational day 15 to postnatal day 10. Growth suppression of offspring was evident with EE especially during the exposure period and was slightly enhanced with the SF diet. On the other hand, most of the female offspring exposed to EE with CRF-1 showed early onset of vaginal opening, strong irregularity in estrous cycle (persistent estrus) and profound histopathological alterations, such as multifollicular ovaries, endometrial hypertrophy, and diffuse hyperplasia of the anterior pituitary. These EE-induced changes were much less pronounced with the SF diet. The results thus demonstrated differential effects of perinatal EE depending on the basal diet used, with enhancement of typical estrogenic responses in females by potential soybean-derived factor(s).
[Show abstract][Hide abstract] ABSTRACT: To evaluate the impact of dietary exposure to endocrine disrupting chemicals (EDCs) during the sensitive period of brain sexual differentiation, maternal Sprague-Dawley rats were fed three representative chemicals, methoxychlor (MXC; 24, 240, and 1200 ppm), genistein (GEN; 20, 200, and 1000 ppm), or diisononyl phthalate (DINP; 400, 4000, and 20,000 ppm), from gestational day 15 to postnatal day 10. Soy-free diet was used as a basal diet to eliminate possible estrogenic effects from the standard diet. Offspring were examined in terms of anogenital distances, prepubertal organ weights, onset of puberty, estrous cyclicity, and organ weights and histopathology of endocrine organs at adult stage (week 11) as well as the volumes of sexually dimorphic nucleus of preoptic area (SDN-POA). All chemicals caused signs of maternal toxicity at high doses. MXC, at 1200 ppm, facilitated and delayed the onset of puberty in females and males, respectively, females also showing endocrine disrupting effects thereafter, such as irregular estrous cyclicity and histopathological alterations in the reproductive tract and anterior pituitary. GEN, at all doses, reduced body weight (BW) at week 11, but did not affect endocrine parameters. Treatment with DINP at 20,000 ppm resulted in degeneration of meiotic spermatocytes and Sertoli cells in the testis and decrease of corpora lutea in the ovary at week 11, although changes remained minimal or slight. The SDN-POA volume remained unchanged with all three chemicals. The results demonstrated that perinatal dietary exposure to EDCs for a limited period causes endocrine disruption in offspring only at high doses.
[Show abstract][Hide abstract] ABSTRACT: We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.
Journal of Histochemistry and Cytochemistry 10/2002; 50(9):1237-45. DOI:10.1177/002215540205000911 · 2.40 Impact Factor