Feasibility study for a downsized comparative thyroid assay with measurement of brain thyroid hormones and histopathology in rats: Case study with 6-propylthiouracil and sodium phenobarbital at high dose.

Concern has been raised that thyroid hormone disruptors (THDs) may potentially interfere with the developing brain, but effects of mild suppression of maternal THs by environmental contaminants on neonatal brain development are not fully understood. The comparative thyroid assay (CTA) is a screening test for offspring THDs, but it requires several animals and is criticized that reliance on serum THs alone as predictive markers of brain malfunction is inadequate. To verify feasibility of the downsized CTA but additional examination of brain THs levels and histopathology, we commenced internal-validation studies. This paper presents the data of the study where 6-propylthiouracil (6-PTU, 10 ppm) and sodium phenobarbital (NaPB, 1000 ppm) were dosed by feeding from gestational days (GD)6-20, and from GD6 to lactation day 21. The modified CTA detected 6-PTU-induced severe (>70%) suppression of serum THs in dams, with >50% suppressed serum/brain TH levels in offspring and brain heterotopia in postnatal day 21 pups. The modified CTA also detected NaPB-induced mild (<35%) suppression of serum THs in dams, with mild (<35%) reduction of serum/brain TH levels in fetuses but not in pups. These findings suggest that the modified CTA may have a potential as a screening test for offspring THDs.

Evaluation of the human hazard of the liver and lung tumors in mice treated with permethrin based on mode of action.

The non-genotoxic synthetic pyrethroid insecticide permethrin produced hepatocellular adenomas and bronchiolo-alveolar adenomas in female CD-1 mice, but not in male CD-1 mice or in female or male Wistar rats. Studies were performed to evaluate possible modes of action (MOAs) for permethrin-induced female CD-1 mouse liver and lung tumor formation. The MOA for liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), increased hepatocellular proliferation, development of altered hepatic foci, and ultimately liver tumors. This MOA is similar to that established for other PPARα activators and is considered to be qualitatively not plausible for humans. The MOA for lung tumor formation by permethrin involves interaction with Club cells, followed by a mitogenic effect resulting in Club cell proliferation, with prolonged administration producing Club cell hyperplasia and subsequently formation of bronchiolo-alveolar adenomas. Although the possibility that permethrin exposure may potentially result in enhancement of Club cell proliferation in humans cannot be completely excluded, there is sufficient information on differences in basic lung anatomy, physiology, metabolism, and biologic behavior of tumors in the general literature to conclude that humans are quantitatively less sensitive to agents that increase Club cell proliferation and lead to tumor formation in mice. The evidence strongly indicates that Club cell mitogens are not likely to lead to increased susceptibility to lung tumor development in humans. Overall, based on MOA evaluation it is concluded that permethrin does not pose a tumorigenic hazard for humans, this conclusion being supported by negative data from permethrin epidemiological studies.

Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

Many nongenotoxic chemicals have been shown to produce liver tumors in mice and/or rats by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with phenobarbital (PB) and other compounds have identified the key events for this MOA: CAR activation; increased hepatocellular proliferation; altered foci formation; and ultimately the development of adenomas/carcinomas. In terms of human relevance, the pivotal species difference is that CAR activators are mitogenic agents in mouse and rat hepatocytes, but they do not stimulate increased hepatocellular proliferation in humans. This conclusion is supported by substantial in vitro studies with cultured rodent and human hepatocytes and also by in vivo studies with chimeric mice with human hepatocytes. Examination of the literature reveals many similarities in the hepatic effects and species differences between activators of rodent CAR and the peroxisome proliferator-activated receptor alpha (PPARα), with PPARα activators also not being mitogenic agents in human hepatocytes. Overall, a critical analysis of the available data demonstrates that the established MOA for rodent liver tumor formation by PB and other CAR activators is qualitatively not plausible for humans. This conclusion is supported by data from several human epidemiology studies.

Application of humanized mice to toxicology studies: Evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR)-mediated mode of action (MOA) for phenobarbital (PB)-induced rodent liver tumor formation has been established, with increased hepatocyte proliferation, which is a key event in tumor formation. Previous studies have demonstrated that PB and other CAR-activators stimulate proliferation in cultured rodent hepatocytes, but not in cultured human hepatocytes. However, in the genetically humanized CAR and pregnane X receptor (PXR) mouse (hCAR/hPXR mouse, downstream genes are still mouse), PB increased hepatocyte proliferation and tumor production in vivo. In contrast to the hCAR/hPXR mouse, studies with chimeric mice with human hepatocytes (PXB-mouse, both receptor and downstream genes are human) demonstrated that PB did not increase human hepatocyte proliferation in vivo. PB increased hepatocyte proliferation in a chimeric mouse model with rat hepatocytes, indicating that the lack of human hepatocyte proliferation is not due to any functional defect in the chimeric mouse liver environment. Gene expression analysis demonstrated that the downstream genes of CAR/PXR activation were similar in hCAR/hPXR and CD-1 mice, but differed from those observed in chimeric mice with human hepatocytes. These findings strongly support the conclusion that the MOA for CAR-mediated rodent liver tumor formation is qualitatively implausible for humans. Indeed, epidemiological studies have found no causal link between PB and human liver tumors. There are many similarities with respect to hepatic effects and species differences between rodent CAR and peroxisome proliferator-activated receptor α activators. Based on our research, the chimeric mouse with human hepatocytes (PXB-mouse) is reliable for human cancer risk assessment of test chemicals.

Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice.

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1-14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Toxicological evaluation of carcinogenicity of the pyrethroid imiprothrin in rats and mice.

The carcinogenic potential of a non-genotoxic pyrethroid imiprothrin was examined in rats and mice. There was no carcinogenicity in rats up to a maximum dose of 5000 ppm of the diet. There was a higher (p = 0.03) incidence of lung adenocarcinomas at 7000 ppm in males, and females showed an increasing (p < 0.01) trend in the incidence of lung adenomas and combined lung adenoma/adenocarcinomas. Additional step sections of lung demonstrated no significant increases in any tumor at p < 0.05, although an increasing trend with dose was observed among females. We argue that, the 7000 ppm dose exceeded the Maximum Tolerated Dose (MTD) for both sexes, based on systemic toxicity as evidenced by body weight gain reduction (both sexes) and high mortality (females). If the 7000 ppm dose is therefore removed from consideration, there are not significant (p < 0.05) increases in tumor formation. Moreover, a consideration of multiple comparisons reveals that the lung tumor increases observed are totally consistent with what would be expected by chance alone. Based on high susceptibility of this mouse strain for the appearance of lung tumors and the lack of a statistically significant increase in tumors by appropriate analysis, the mouse study does not indicate a carcinogenic effect of imiprothrin, and thus no classification for carcinogenicity is warranted.

Effect of the anatomical site on telomere length and pref-1 gene expression in bovine adipose tissues.

Adipose tissue growth is associated with preadipocyte proliferation and differentiation. Telomere length is a biological marker for cell proliferation. Preadipocyte factor-1 (pref-1) is specifically expressed in preadipocytes and acts as a molecular gatekeeper of adipogenesis. In the present study, we investigated the fat depot-specific differences in telomere length and pref-1 gene expression in various anatomical sites (subcutaneous, intramuscular and visceral) of fattening Wagyu cattle. Visceral adipose tissue expressed higher pref-1 mRNA than did subcutaneous and intramuscular adipose tissues. The telomere length in visceral adipose tissue tended to be longer than that of subcutaneous and intramuscular adipose tissues. The telomere length of adipose tissue was not associated with adipocyte size from three anatomical sites. No significant correlation was found between the pref-1 mRNA level and the subcutaneous adipocyte size. In contrast, the pref-1 mRNA level was negatively correlated with the intramuscular and visceral adipocyte size. These results suggest that anatomical sites of adipose tissue affect the telomere length and expression pattern of the pref-1 gene in a fat depot-specific manner.

Fat depot-specific differences in pref-1 gene expression and adipocyte cellularity between Wagyu and Holstein cattle.

Preadipocyte factor-1 (pref-1) is specifically expressed in preadipocytes and acts as a gatekeeper of adipogenesis by maintaining the preadipocyte state and preventing adipocyte differentiation. We hypothesized that the breed differences of adipogenic capacity in cattle could be explained by the expression level of pref-1. In this experiment, we studied the expression level of the pref-1 gene and adipocyte cellularity in subcutaneous and mesenteric adipose tissues of Japanese Black (Wagyu) and Holstein fattening cattle. In subcutaneous adipose tissue, there were no significant differences in the pref-1 gene expression levels and adipocyte sizes between the breeds. In contrast, the expression level of the pref-1 gene in mesenteric adipose tissue of Holsteins was significantly higher than that of Wagyu. In addition, the size of mesenteric adipocytes in Holsteins was significantly smaller than that of Wagyu. These results indicate that the breed differences of fattening cattle affect the expression pattern of the pref-1 gene and adipocyte cellularity in a fat depot-specific manner.

Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital.

The Comparative Thyroid Assay (CTA, USEPA) is a screening test for thyroid hormone (TH) disruption in peripheral blood of dams and offspring. Recently, we began investigating feasible improvements to the CTA by adding examination of offspring brain TH concentrations and brain histopathology. In addition, we hypothesize that the number of animals required could be reduced by 50 % while still maintaining sensitivity to characterize treatment related changes in THs. Previously, we showed that the prenatal test cohort of the modified CTA could detect 1000 ppm sodium phenobarbital (NaPB)-induced suppression of brain T3 (by 9 %) and T4 (by 33 %) with no significant changes in serum T3 and T4 (less than 8 %). In the current study we expanded the dose response in a prenatal test cohort. Pregnant SD rats (N = 10/group) were exposed to 0, 1000 or 1500 ppm NaPB in the diet from gestational days (GD) 6 to GD20. Serum THs concentrations in GD20 dams together with serum/brain THs concentrations and brain histopathology in the GD20 fetuses were examined. NaPB dose-dependently suppressed serum T3 (up to -26 %) and T4 (up to -44 %) in dams, with suppression of T3 in serum (up to -26 %) and brain (up to -18 %) and T4 in serum (up to -26 %) and brain (up to -29 %) of fetuses but without clear dose dependency. There were no remarkable findings that deviated significantly from controls in GD20 fetal brain by qualitative histopathology. Overall, the present study suggests that the prenatal test cohort of this modified CTA is able to detect the expected fetal TH disruptions by prenatal exposure to NaPB, while also reducing the number of animals used by 50 %, consistent with the results of our previous study. These findings add to the suggestion that lowering group sizes and adding endpoints may be a useful alternative to the original CTA design.

Double Primary Cancer of the Prostate and Urothelial Cancer: A Single Institution Experience.

Prostate cancer (PCa) ranks as the second most common cancer in Japanese males, while bladder cancer (BC) holds the tenth spot. Among double urological cancers, the incidence of synchronous or metachronous BC and PCa is the highest. Reports on upper urinary tract (UUT) urothelial cancer (UC) in PCa patients are limited. Here, we present three cases of metachronous PCa and BC, with subsequent diagnosis of ureteral and renal pelvic cancer during the course of the disease. In the follow-up of patients with urological cancers, it is important to be aware not only of the progression of the initial cancer but also the potential development of a second cancer.

Diet-induced changes in Ucp1 expression in bovine adipose tissues.

Brown adipocytes, which regulate non-shivering thermogenesis, have been believed to exist in a limited number of mammalian species, and only under limited physiological conditions. Recent discoveries indicate that adult humans possess a significant number of functional brown adipocytes. This study explores the regulatory emergence of brown adipocytes in white adipose tissue (WAT) depots of fattening cattle. RT-PCR analyses indicated significant expression of Ucp1, a brown adipocyte-specific gene, in the WAT of 31-month-old Japanese Black steers. Immunohistochemical analysis revealed that Ucp1-positive small adipocytes were dispersed in the subcutaneous WAT. Next, we examined expression level of Ucp1 and other brown adipocyte-selective genes such as Pgc1α, Cidea, Dio2, Cox1, Cox7a1 and Cox8b in WAT of 30-month-old steers fed either diet with low protein/energy content (roughage diet) or that with high protein/energy content (concentrate diet) for 20months. Ucp1 expression in the subcutaneous WAT was significantly higher in the concentrate diet group than in the roughage diet group. Furthermore, the higher Ucp1 expression levels were limited to the subcutaneous WAT, and no differences between groups were detected in the mesenteric, perirenal, intermuscular or intramuscular WAT. Expression of Dio2, Cox1 and Cox8b was higher in the subcutaneous WAT but not in the mesenteric WAT of the concentrate diet group. Furthermore, expression of Prdm16, a positive regulator of differentiation toward brown adipocyte-lineage cells, and expression of leptin, a molecule that enhances activity of brown adipocytes, were significantly higher in the subcutaneous WAT of the concentrate diet group. This study demonstrates the presence of brown adipocytes in WAT depots of fattening cattle, and suggests the diet-related modulation of expression of genes predominantly expressed in brown adipocytes.

Prediction of methane emissions from fattening cattle using the methane-to-carbon dioxide ratio.

This study aimed to develop a prediction equation for methane (CH4 ) emissions from fattening cattle based on the CH4 /carbon dioxide (CO2 ) ratio and validate the predictive ability of the developed equation. The prediction equation was developed using the CH4 /CO2 ratio combined with oxygen consumption and respiratory quotient estimations that were theoretically calculated from the relation between gas emissions and energy metabolism. To validate the prediction equation, gas measurements in the headboxes were conducted using eight Japanese Black steers. The predictive ability of the developed equation was compared with that of two previously reported equations. As a result, the developed and reported equations had significant (P < 0.01) linear relationships between the observed and predicted CH4 emissions. Notably, only the developed equation had a significant (P < 0.01) linear relationship between the observed and predicted CH4 emissions when expressed per unit of dry matter intake. The results suggest that the developed prediction equation has a higher predictive ability than previously reported equations, particularly in evaluating the efficiency of CH4 emissions. Although further validation is required, the equation developed in this study can be a valuable tool for on-farm estimations of individual CH4 emissions from fattening cattle.

Mammal toxicology of synthetic pyrethroids.

Pyrethroids show moderate acute oral toxicity in rodents, and their typical toxicological signs are tremors (T syndrome) for Type I (generally non-cyano pyrethroids) and choreoathetosis with salivation (CS syndrome) for Type II (generally α-cyano pyrethroids). However, some pyrethroids show mixed clinical signs. Mainly Type II pyrethroids cause paresthesia, which is characterized by transient burning/tingling/itching sensation of the exposed skin. Also, it has been suggested that some pyrethroids cause developmental neurotoxicity, but available evidence has been judged to be insufficient. While some pyrethroids have been shown to cause tumors in rodent models, the tumor induction does not appear to reflect a common carcinogenic endpoint for this particular subset of compounds. Analysis of carcinogenic mode of action in some cases provides evidence not relevant in humans. Pyrethroids produce no common teratogenic effects in a particular species based on similarity in structure or mode of insecticidal action.

Intramuscular adipogenesis in cattle: Effects of body fat distribution and macrophage infiltration.

Ectopic fat is defined by the deposition of adipose tissue within non-adipose tissue such as skeletal muscle. Japanese Black cattle (Wagyu) are characterized by the ability to accumulate high amounts of intramuscular adipose tissue. Obese conditions enhance the accumulation of ectopic fat. This review shows the effects of subcutaneous and visceral fat distribution on Wagyu intramuscular adipogenesis. Obese conditions also stimulate the macrophage infiltration into adipose tissues. Adipose tissue macrophages have reported to regulate adipose tissue growth and ectopic fat accumulation in humans and rodents. Wagyu is characterized by the higher capacity for intramuscular adipogenesis than Holsteins. This review discusses the depot-specific effects of macrophage infiltration among subcutaneous, visceral, and intramuscular adipose tissue on intramuscular adipogenesis in Wagyu and Holstein cattle. Recently, metabolome analysis has been used to identify obesity-related biomarkers by comparing the biological samples between lean and obese patients. This review introduces the metabolomic profiles of plasma and intramuscular adipose tissue between Wagyu and Holsteins.

Metabolomic analysis of plasma and intramuscular adipose tissue between Wagyu and Holstein cattle.

In this experiment, we studied the effects of breed differences in intramuscular adipogenic capacity on the metabolomic profiles of plasma and intramuscular adipose tissue between Wagyu (high intramuscular adipogenic capacity) and Holstein (low intramuscular adipogenic capacity) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the intramuscular fat content, intramuscular adipocyte size and the expression of adipogenic transcription factors (C/EBPß and C/EBPα) of Wagyu were significantly higher than those of Holstein. Metabolites detected at significantly higher levels in Wagyu plasma were related to the tricarboxylic acid cycle, lipid synthesis, fatty acid metabolism, diabetes, and glucose homeostasis. In contrast, metabolites detected at significantly higher levels in Holstein plasma were related to choline metabolism, the ethanolamine pathway, glutathione homeostasis, nucleic acid metabolism, and amino acid metabolism. Metabolites detected at significantly higher levels in Holstein intramuscular adipose tissue were related to nucleic acid metabolism, amino acid metabolism, amino sugar metabolism, beta oxidation, and the ethanolamine pathway. There were no metabolites significantly higher levels in Wagyu intramuscular adipose tissue. These results indicate candidate biomarkers of breed differences in intramuscular adipogenic capacity between Wagyu and Holstein.

Well-differentiated teratoma in a mouse uterus.

Teratomas commonly occur in the testis and ovary, whereas in the uterus they are rare. The authors report findings for a mass detected in the uterus of a 26-week-old mouse in a colony of C57BL/6 bred in their laboratory. The mass was located in the endometrium and protruded into the lumen. Histopathologically, it consisted of abnormal diploblastic or triploblastic tissues. Bone with a growth plate and myeloid cells, as well as cartilage, was mainly observed. It also included melanocytes, exocrine gland-like cells, striated muscle, and neuron-like cells. While these tissues were accompanied by extensive necrosis, all of them were well differentiated and lacked features of malignancy, such as invasion and metastasis. This mouse had experienced parturition, but fetal tissue was not observed in the lesion. Therefore, the lesion was diagnosed as a benign teratoma, which was spontaneously developed in the uterus.

Effects of low-crude protein diets supplemented with rumen-protected lysine and methionine on fattening performance and nitrogen excretion of Holstein steers.

The objective of this experiment was to investigate the effects of low-crude protein (CP) diets supplemented with rumen-protected lysine and methionine on growth performance, nitrogen excretion, and carcass traits in Holstein steers. Steers consumed the following diets: (1) 17.2% CP on a dry-matter basis during the early period (from 7 to 10 months of age) and 14.5% CP during the late period (from 10 to 18 months of age; CON, n = 4, initial body weight [BW] 238 kg), and (2) 14.4% CP during the early period and 11.4% CP during the late period (AA, n = 4, initial BW 243 kg). The AA diet contains rumen-protected lysine and methionine. Except for CP intake, feed intake and body weight gain were not affected by dietary CP content. Total nitrogen excretion per metabolic BW tended to be lower (p < .10) in the early period and significantly lower (p < .05) in the late period with decreasing the feed CP content. Plasma urea nitrogen concentrations were lower in AA than CON. Carcass traits and total free amino acid contents of the longissimus thoracis muscle were not affected by dietary CP content. Adding rumen-protected lysine and methionine to a low-CP diet would reduce nitrogen excretion in fattening Holstein steers without affecting productivity.

Club Cells Are the Primary Target for Permethrin-Induced Mouse Lung Tumor Formation.

Permethrin has been shown to increase lung adenomas in female CD-1 mice, but not in male mice or Wistar rats. The proposed mode of action (MOA) for permethrin-induced female mouse lung tumor formation involves morphological changes in Club cells; increased Club cell proliferation; increased Club cell hyperplasia, and lung tumor formation. In this study, the treatment of female CD-1 mice with tumorigenic doses (2500 and 5000 ppm) of permethrin, but not with a nontumorigenic dose (20 ppm), for 14 and/or 28 days increased Club cell replicative DNA synthesis. Global gene expression analysis of female mouse lung samples demonstrated that permethrin treatment up-regulated 3 genes associated with cell proliferation, namely aldehyde dehydrogenase 3a1 (Aldh3a1), oxidative stress-induced growth inhibitor 1, and thioredoxin reductase 1. Treatment with 2500 and 5000 ppm, but not 20 ppm, permethrin for 7 days produced significant increases in mRNA levels of these 3 genes. Immunohistochemical analysis demonstrated that Club cell secretory protein, CYP2F2, and ALDH3A1 colocalized in Club cells; confirmed by flow cytometry analysis of lung cells employing KI67 as a cell proliferation marker. Overall, the present data extend the proposed MOA by demonstrating that Club cells are the primary initial target of permethrin administration in female mouse lungs. As humans are quantitatively much less sensitive to agents that increase Club cell proliferation and lung tumor formation in mice, it is most likely that permethrin could not produce lung tumors in humans. This conclusion is supported by available negative epidemiological data from several studies.

Fat depot-specific differences in angiogenic growth factor gene expression and its relation to adipocyte size in cattle.

Adipocytes derived from different anatomical sites vary in the expression of adipocytokines and growth factor genes. Adipogenesis is tightly associated with angiogenesis, although the regional variation of angiogenic growth factor gene expression in adipose tissues remains unclear. In this experiment, we studied the fat depot-specific differences (subcutaneous, intramuscular, intermuscular, renal, and mesenteric) in the expression of angiogenic growth factor mRNA [vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), and leptin], as well as the relationship between angiogenic growth factor mRNA level and adipocyte size in bovine adipose tissues. Intermuscular, renal, and mesenteric adipose tissues expressed significantly higher VEGF, FGF-2, and leptin mRNA levels than did subcutaneous and intramuscular adipose tissues. Mesenteric adipose tissue also expressed higher FGF-10 mRNA levels than did subcutaneous and intramuscular adipose tissues. There was no significant difference in the expression of HGF mRNA among adipose tissue depots. A significant correlation existed between adipocyte size and VEGF, FGF-2, FGF-10, and leptin mRNA levels. These results indicate that fat depot-specific difference in angiogenic growth factor gene expression results from the difference in adipocyte size.