Feeding Studies In Mice Research Articles

Mouse feeding study and microbiome analysis of sourdough bread for evaluation of its health effects.

Sourdough bread fermented with yeast and lactic acid bacteria (LAB) is thought to have various beneficial health effects. However, its beneficial effects were not fully evaluated with in vivo mouse model. To evaluate these effects in vivo, a mouse feeding study and microbiome analysis of white bread containing 40% sourdough (WBS) and yeast-leavened white bread (WB) were performed. Although feed consumption and body weight increased with WBS, the glycemic index was reduced, suggesting a diabetes-lowering effect, probably due to the presence of dietary fiber and short-chain fatty acids (SCFA). In addition, a mineral absorption test showed that WBS increased magnesium absorption owing to phytate degradation during fermentation. Interestingly, WBS decreased total cholesterol and triglycerides, probably due to the dietary fiber and SCFA in LAB. In addition, the ratio of low- and high-density lipoprotein was decreased in WBS, implying potential risk reduction for cardiovascular disease. An immunomodulatory assay of WBS revealed that pro-inflammatory cytokines TNF-α and IL-6 were decreased, suggesting anti-inflammatory activity. Gluten degradation by fermentation and antioxidation activity of menaquinol/ubiquinol by gut microbiota also supported the anti-inflammatory activity of sourdough bread. Furthermore, some beneficial gut bacteria, including Akkermansia, Bifidobacterium, and Lactobacillus, were increased in WBS. In particular, Akkermansia has been associated with anti-inflammatory properties. Consequently, WBS has beneficial effects on health, including decreased glycemic index and cholesterol, increased mineral availability and absorption, anti-inflammatory properties, and establishment of healthy gut microbiota.

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms

IntroductionA potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are...

Dietary Supplementation with Fermented Brassica rapa L. Stimulates Defecation Accompanying Change in Colonic Bacterial Community Structure.

Our previous studies have elucidated that oral administration of Brassica rapa L. extract, known as Nozawana in Japan, alters immune responses and gut microbiota composition, increasing the numbers of butyrate-producing bacteria. Therefore, further investigation would help elucidate the mechanism attributable for the changes and health-promoting effects observed after B rapa L. extract ingestion. To reveal the modulation effects of fermented B. rapa L. on immune function and intestinal bacterial community structure, we conducted an intervention study with healthy volunteers followed by a mouse feeding study. The pilot intervention study was conducted for healthy volunteers aged 40–64 years under the hypothesis that the number of subjects exhibiting any change in gut microbiota in response to fermented B. rapa L. consumption may be limited. In total, 20 volunteers consumed 30 g of fermented B. rapa L. per day for 4 weeks. The fecal bacterial community composition of the volunteers was characterized using terminal-restriction fragment length polymorphism patterning followed by clustering analysis. To evaluate the detailed changes in the immune responses and the gut bacterial composition, assessed by high-throughput sequencing, we fed healthy mice with freeze-dried, fermented B. rapa L. for 2 weeks. The fecal bacterial community composition of the volunteers before the intervention was divided into three clades. Regardless of the clade, the defecation frequency significantly increased during the intervention weeks compared with that before the intervention. However, this clustering detected a specific increase of Prevotella in one cluster (low to zero Prevotella and high occupation of Clostridium at clusters IV and XIVa) post-ingestion. The cytokine production of spleen cells significantly increased due to feeding fermented B. rapa L. to the mice. This supplementary in vivo trial provided comparable results to the volunteer study regarding the effects of ingestion of the material given the compositional change complying with that of dietary fiber, particularly in the increase of genera Prevotella, Lachnospira, and genera in the Ruminococcaceae family, and the increase in daily defecation amount during 2 weeks of administration. We conclude that feeding fermented B. rapa L. may be responsible for the observed modulation in gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria which may be relevant to the improvement in bowel function such as defecation frequency.

Delineating effect of corn microRNAs and matrix, ingested as whole food, on gut microbiota in a rodent model.

Dietary microRNAs (miRNAs) are thought to regulate a wide range of biological processes, including the gut microbiota. However, it is difficult to separate specific effect(s) of miRNA from that of the food matrix. This study aims to elucidate the specific effect(s) of dietary corn miRNAs, ingested as a whole food, on the gut microbiota. We developed an autoclave procedure to remove 98% of miRNA from corn. A mouse feeding study was conducted comparing autoclaved corn to nonautoclaved corn and purified corn miRNA. Compared to nonspecific nucleotides and corn devoid of miRNAs, feeding purified corn miRNAs or corn to C57BL/6 mice via gavage or diet supplementation for two weeks lead to a decrease in total bacteria in the cecum. The effect appeared to be due to changes in Firmicutes. Additionally, corn matrix minus miRNA and processing also affected gut bacteria. In silico analysis identified corn miRNAs that aligned to Firmicutes genome sequences lending further support to the interaction between corn miRNAs and this bacterium. These data support interactions between plant food miRNA, as well as matrix, and the gut microbiota exist but complex. However, it provides additional support for mechanism by which bioactive dietary components interact with the gut microbiota.

High Dietary Kuding Tea Extract Supplementation Induces Hepatic Xenobiotic-Metabolizing Enzymes-A 6-Week Feeding Study in Mice.

Kuding tea (KT) is a traditional Chinese beverage rich in plant bioactives that may exhibit various health benefits. However, little is known about the safety of KT extract (KTE) when consumed long term at high doses as a dietary supplement. Therefore, in this study, we investigated aspects of the safety of KTE. Male C57BL/6 mice were fed a high-fat, high-fructose, Western-type diet (control) supplemented with either 12.88% γ-cyclodextrin (γCD), 7.12% KTE (comprising 0.15% ursolic acid, UA) encapsulated in 12.88% γCD (KTE-γCD), or 0.15% UA over a 6-week experimental period. The dietary treatments did not affect food intake, body weight or body composition. However, treatment with KTE-γCD, but not γCD and UA, increased liver weight and hepatic fat accumulation, which was accompanied by increased hepatic PPARγ and CD36 mRNA levels. KTE-γCD treatment elevated plasma cholesterol and CYP7A1 mRNA and protein levels compared to those in control mice. KTE-γCD substantially increased the mRNA and protein levels of hepatic CYP3A and GSTA1, which are central to the detoxification of drugs and xenobiotics. Furthermore, we observed a moderate elevation in hepatic CYP3A (5-fold change) and GSTA1 (1.7-fold change) mRNA levels in UA-fed mice. In vitro data collected in HepG2 cells indicated a dose-dependent increase in hepatic cytotoxicity in response to KTE treatment, which may have been partly mediated by UA. Overall, the present data may contribute to the safety assessment of KTE and suggest that KTE encapsulated in γCD affects liver fat storage and the hepatic phase I and phase II responses in mice.

Acute and 28-day repeated dose toxicity evaluations of 2-hydroxybenzylamine acetate in mice and rats

2-hydroxybenzylamine (2-HOBA), a compound naturally found in buckwheat, has been shown to protect cells and tissues from the damaging effects of oxidative stress. The purpose of this report was to evaluate 2-HOBA in preclinical oral rodent toxicity studies. This report includes the results from three oral toxicity studies in rodents: a preliminary 28-day feeding study in mice, a 14-day acute oral toxicity study in rats, and a 28-day repeated dose oral toxicity study in rats. The preliminary mouse feeding study showed no adverse effects of 2-HOBA at concentrations up to 0.456% by weight in feed, but decreased food intake and weight loss were observed at 1.56% 2-HOBA in the diet, likely due to poor palatability. In the acute dosing study, 2000 mg/kg BW 2-HOBA resulted in mortality in one of the six tested female rats, indicating a median lethal dose of 2500 mg/kg BW. In the 28-day repeated oral dose study, small differences were observed between 2-HOBA treated and control group rats, but none of these differences were determined to be of toxicological significance. Together, these studies support the lack of toxicity of oral administration of 2-HOBA acetate at doses up to 1000 mg/kg BW d−1 in rodents.

Metabolic Effects of High Glycaemic Index Diets: A Systematic Review and Meta-Analysis of Feeding Studies in Mice and Rats.

Low glycaemic index (LGI) diets are often reported to benefit metabolic health, but the mechanism(s) responsible are not clear. This review aimed to systematically identify studies investigating metabolic effects of high glycaemic index (HGI) versus LGI diets in mice and rats. A meta-analysis was conducted to calculate an overall effect size, Hedge’s standardised mean differences (hereafter d), for each trait, with moderator variables considered in subsequent meta-regressions. Across 30 articles, a HGI diet increased five of the seven traits examined: body weight (d = 0.55; 95% confidence interval: 0.31, 0.79), fat mass (d = 1.08; 0.67, 1.49), fasting circulating insulin levels (d = 0.40; 0.09, 0.71), and glucose (d = 0.80; 0.35, 1.25) and insulin (d = 1.14; 0.50, 1.77) area under the curve during a glucose tolerance test. However, there was substantial heterogeneity among the effects for all traits and the small number of studies enabled only limited investigation of possible confounding factors. HGI diets favour body weight gain, increased adiposity and detrimentally affect parameters of glucose homeostasis in mice and rats, but these effects may not be a direct result of GI per se; rather they may be due to variation in other dietary constituents, such as dietary fibre, a factor which is known to reduce the GI of food and promote health via GI-independent mechanisms.

Consumption of antimicrobial manuka honey does not significantly perturb the microbiota in the hind gut of mice.

The aim of this study was to test the hypothesis that consuming manuka honey, which contains antimicrobial methylglyoxal, may affect the gut microbiota. We undertook a mouse feeding study to investigate whether dietary manuka honey supplementation altered microbial numbers and their production of organic acid products from carbohydrate fermentation, which are markers of gut microbiota function. The caecum of C57BL/6 mice fed a diet supplemented with antimicrobial UMF® 20+ manuka honey at 2.2 g/kg animal did not show any significantly changed concentrations of microbial short chain fatty acids as measured by gas chromatography, except for increased formate and lowered succinate organic acid concentrations, compared to mice fed a control diet. There was no change in succinate-producing Bacteroidetes numbers, or honey-utilising Bifidobacteria, nor any other microbes measured by real time quantitative PCR. These results suggest that, despite the antimicrobial activity of the original honey, consumption of manuka honey only mildly affects substrate metabolism by the gut microbiota.

High‐Iron Intake Impairs Growth and Causes Copper‐Deficiency Anemia in Mice

A commonly used method to cause iron loading in rodents is feeding of a 1–2% carbonyl iron diet. This high amount of dietary iron presumably bypasses normal protective mechanisms and leads to increased body iron levels. We utilized this dietary iron‐overload model in a mouse feeding study in which we also altered the dietary copper content. This study was intended to test the hypothesis that dietary copper would influence intestinal iron absorption during iron overload. Weanling, male C57BL/6 mice were housed in overhanging, wire mesh‐bottomed cages and fed one of 6 different AIN‐93G‐based diets for 4 weeks containing normal (79 ppm) or high (8820 ppm) iron in combination with low (0.9 ppm), normal (8 ppm) or high (183 ppm) copper. Diets were otherwise identical. All data reported below were from 4–8 mice per group and all reached statistical significance (p<0.05; 2–factor ANOVA). To our surprise, mice fed the high‐iron diets with low and normal copper levels grew slower and were severely anemic, but the higher copper content normalized growth rate and corrected the anemia. Transferrin saturation, hepatic non‐heme iron content, and hepatic hepcidin mRNA expression were all uniformly elevated in all groups of mice fed the high‐iron diets. We also noted decreased heart copper content and cardiac hypertrophy in the mice fed the high‐iron diet with low and normal copper, but the high‐copper diet corrected this defect. Moreover, hepatic copper content was lower and serum ceruloplasmin activity was depressed in the high‐iron fed mice with the low and normal copper content, but both were restored to control levels in the group fed the high‐iron diet with high copper. These findings suggested that the high dietary iron intake with low and normal copper caused severe copper‐deficiency anemia. We postulated that the high‐iron content blocked intestinal copper absorption, which then resulted in systemic copper deficiency. We thus tested this hypothesis by performing in vivo intestinal copper absorption studies. Mice were again fed the same diets for 4 weeks, fasted overnight and 64Cu was then administered to the mice by oral gavage. Mice were immediately given ad libitum access to the same diets and then sacrificed ~8 hours later. This time point ensured that no copper was excreted in the feces since transit time in mice is ~11 hours. This is important since copper can be excreted in the bile via the feces. Copper absorption was calculated as a ratio of counts in the carcass (minus the GI tract) to total counts administered by gavage × 100. Results showed that mice fed the low copper, adequate‐iron diet had higher copper absorption (>60% of administered dose). Mice fed the low‐copper diet with high iron, however, failed to upregulate copper absorption. Further, although mice fed the adequate copper, high‐iron diet, suffered from copper‐deficiency anemia, they did not have the ability to upregulate copper absorption. Additionally, 64Cu accumulation in all tissues tested was significantly lower than in the low‐copper, adequate‐iron fed group. Collectively, these data then suggest that high‐body iron levels impair intestinal copper absorption and perturb copper distribution, providing further evidence of physiologically relevant iron‐copper interactions in mammals.Support or Funding InformationNIH grant 1R01 DK074867 (to J.F.C.) Diet iron and copper concentration: For the iron and copper interaction study, the diets were fabricated based on the AIN‐93G formulation (Dyets Inc., Bethlehem). Adequate, and high‐iron diets contained 79, 8820 ppm iron, respectively. Low, adequate, and high‐copper diets contained 0.9, 8, 183 ppm copper, respectively. Each 3 iron and copper contents were used to formulate the 9 different diets. The diets were otherwise identical. Fe(ppm) Cu(ppm) FeACuD 93.7 0.92 FeACuA 71.9 8.96 FeACuE 71.8 183 FeECuD 9036 0.94 FeECuA 8707 9.18 FeECuE 8718 184

Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo

Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3 ± 11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P ≤ 0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (P < 0.02) in mice fed anti-m16 (1% as dried egg yolk powder) and 30% (P < 0.0001) in mice fed sevelamer HCl (1% of diet) when compared to mice fed nonspecific egg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases.

Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential

BackgroundBradycardic agents are of interest for the treatment of ischemic heart disease and heart failure, as heart rate is an important determinant of myocardial oxygen consumption.ObjectivesThe purpose of this study was to investigate the propensity of hydroxychloroquine (HCQ) to cause bradycardia.MethodsWe assessed the effects of HCQ on (1) cardiac beating rate in vitro (mice); (2) the “funny” current (If) in isolated guinea pig sinoatrial node (SAN) myocytes (1, 3, 10 µM); (3) heart rate and blood pressure in vivo by acute bolus injection (rat, dose range 1–30 mg/kg), (4) blood pressure and ventricular function during feeding (mouse, 100 mg/kg/d for 2 wk, tail cuff plethysmography, anesthetized echocardiography).ResultsIn mouse atria, spontaneous beating rate was significantly (P < .05) reduced (by 9% ± 3% and 15% ± 2% at 3 and 10 µM HCQ, n = 7). In guinea pig isolated SAN cells, HCQ conferred a significant reduction in spontaneous action potential firing rate (17% ± 6%, 1 μM dose) and a dose-dependent reduction in If (13% ± 3% at 1 µM; 19% ± 2% at 3 µM). Effects were also observed on L-type calcium ion current (ICaL) (12% ± 4% reduction) and rapid delayed rectifier potassium current (IKr) (35% ± 4%) at 3 µM. Intravenous HCQ decreased heart rate in anesthetized rats (14.3% ± 1.1% at 15mg/kg; n = 6) without significantly reducing mean arterial blood pressure. In vivo feeding studies in mice showed no significant change in systolic blood pressure nor left ventricular function.ConclusionsWe have shown that HCQ acts as a bradycardic agent in SAN cells, in atrial preparations, and in vivo. HCQ slows the rate of spontaneous action potential firing in the SAN through multichannel inhibition, including that of If.

R-α lipoic acid γ-cyclodextrin complex increases energy expenditure: A 4-month feeding study in mice

ObjectiveA high-fat diet (HFD) affects energy expenditure in laboratory rodents. R-α lipoic acid cyclodextrin (RALA-CD) complex is a stable form of lipoic acid (LA) and may improve energy expenditure. The aim of this study was to determine the effect of RALA-CD on energy expenditure and underlying molecular targets in female laboratory mice. MethodsFemale C57BL/6J mice were fed a HFD containing 0.1% LA for about 16 wk. The effects on energy expenditure, gene and protein expression were assessed using indirect calorimetry, real-time reverse transcriptase polymerase chain reaction, and Western blot, respectively. ResultsSupplementing mice with RALA-CD resulted in a significant increase in energy expenditure. However, both RALA per se (without γ-cyclodextrin) and S-α lipoic acid cyclodextrin did not significantly alter energy expenditure. Furthermore RALA-CD changed expression of genes encoding proteins centrally involved in energy metabolism. Transcriptional key regulators sirtuin 3 and peroxisome proliferator-activated receptor-γ, coactivator 1 alpha, as well as thyroid related enzyme type 2 iodothyronine deiodinase were up-regulated in brown adipose tissue (BAT) of RALA-CD–fed mice. Importantly, mRNA and/or protein expression of downstream effectors uncoupling protein (Ucp) 1 and 3 also were elevated in BAT from RALA-CD-supplemented mice. ConclusionOverall, present data suggest that RALA-CD is a regulator of energy expenditure in laboratory mice.

Biotinylation of the c‐myc promoter binding protein MBP‐1 decreases c‐myc expression in mammary carcinoma MCF‐7 cells

Biotin feeding studies in mice predisposed to developing breast cancer suggest that the tumor load increases by ~100% in biotin‐deficient mice compared with controls, and that the increase is associated with metastasis rather than new tumors. The oncogene c‐myc is implicated in cancer metastasis, and its expression is repressed by binding of MBP‐1 to the c‐myc promoter. Discovery‐based studies for identifying novel biotin‐containing proteins suggest that MBP‐1 contains covalently bound biotin, judged by mass spectrometry. We hypothesized that biotinylation of MBP‐1 increases the binding of MBP‐1 to the c‐myc promoter, thereby decreasing c‐myc expression and metastasis. MCF‐7 cells were cultured in biotin‐defined media (deficient, sufficient, supplemented) and transfected with a reporter gene construct for the c‐myc promoter. Reporter activity was ~100% greater in biotin‐deficient cells compared with sufficient and supplemented controls. Likewise, c‐myc mRNA levels were 10% higher in deficient compared with sufficient cells, and 10% high in sufficient compared with supplemented cells. Biotinylation of MBP‐1 is an enzymatic process, judged by streptavidin blots. These observations are consistent with our hypothesis and preliminary studies, suggesting that biotin depletion causes an increased expression of the oncogene c‐myc, thereby increasing cancer risk.Grant Funding Source: ARD Hatch, NIFA, and NIH

Dietary omega-3 PUFA and health: Stearidonic acid-containing seed oils as effective and sustainable alternatives to traditional marine oils

The daily consumption of dietary omega-3 PUFA is recommended by governmental agencies in several countries and by a number of health organizations. The molecular mechanisms by which these dietary PUFA affect health involve the enrichment of cellular membranes with long-chain 20- and 22-carbon omega-3 PUFA that impacts tissues by altering membrane protein functions, cell signaling, and gene expression profiles. These changes are recognized to have health benefits in humans, especially relating to cardiovascular outcomes. Cellular membrane enrichment and health benefits are associated with the consumption of long-chain omega-3 PUFA found in marine oils, but are not generally linked with the consumption of alpha-linolenic acid, the 18-carbon omega-3 PUFA found in plant seed oils. However, the supply of omega-3 PUFA from marine sources is limited and may not be sustainable. New plant-derived sources of omega-3 PUFA like stearidonic acid-soy oil from genetically modified soybeans and Ahiflower oil from Buglossoides arvensis seeds that are enriched in the 18-carbon omega-3 PUFA stearidonic acid are being developed and show promise to become effective as well as sustainable sources of omega-3 PUFA. An example of changes in tissue lipid profiles associated with the consumption of Ahiflower oil is presented in a mouse feeding study.

Modulation of cholesterol metabolism by Ginkgo biloba L. nuts and their extract

Ginkgo biloba L. is among the oldest species of trees on earth. Various parts of the tree have a history of use in prevention of cardiovascular and neurodegenerative diseases. However, most of the current scientific research is focused on the Ginkgo leaf extract. Thus, we tested the effect of Ginkgo nuts on lipid metabolism as a therapeutic for cardiovascular diseases. Four preparations of Ginkgo nuts (whole nuts, their methanol extract and its lipid and water soluble fractions) were tested using Human Hepatoma cells (Hep G2) and in vivo mouse feeding study. Ginkgo nut preparation may modulate serum cholesterol levels by modulating Apolipoprotein B secretion as well as low-density lipoprotein receptor in the liver. From mouse feeding trial, Ginkgo nut supplementation, as whole nuts, reduced the levels of hepatic cholesterol while increasing serum cholesterol compared to control. The lipid soluble fraction was responsible for decreased hepatic cholesterol, while the water soluble fraction may contribute to increased serum cholesterol. The lipid soluble fraction of Ginkgo nut may have potential to be used for the purpose of prevention of cardiovascular diseases. This is the first report investigating the effects of Ginkgo nuts on cholesterol metabolism.

The presence of drug in control samples during toxicokinetic investigations—A Novartis perspective

During a submission procedure, the validity of a few dietary toxicity studies was questioned because low levels of the drug were detected among control toxicokinetic samples. Although several lines of reasoning suggested that these findings arose from ex vivo contamination, the Regulatory Authority stated that it was not possible to establish a no-effect-level in any of the studies and so the submission was withdrawn. In response, Novartis conducted a thorough review and modification of the procedures involved in the collection and analysis of toxicokinetic samples to minimize such contamination in future studies. Ongoing monitoring of contamination in toxicology studies has subsequently revealed that although it was not possible to completely eliminate the problem, the new procedures together with an increasing awareness of the issue have considerably reduced the incidence of contamination. The process of contamination and its control was also modeled in a feeding study in mice. This provided good evidence that the detection of drug in control samples in the previous studies originated from external sources and not from in vivo exposure.

Thiamethoxam Induced Mouse Liver Tumors and Their Relevance to Humans

Thiamethoxam, a neonicotinoid insecticide, which is not mutagenic either in vitro or in vivo, caused an increased incidence of liver tumors in mice when fed in the diet for 18 months at concentrations in the range 500 to 2500 ppm. A number of dietary studies of up to 50 weeks duration have been conducted in order to identify the mode of action for the development of the liver tumors seen at the end of the cancer bioassay. Both thiamethoxam and its major metabolites have been tested in these studies. Over the duration of a 50-week thiamethoxam dietary feeding study in mice, the earliest change, within one week, is a marked reduction (by up to 40%) in plasma cholesterol. This was followed 10 weeks later by evidence of liver toxicity including single cell necrosis and an increase in apoptosis. After 20 weeks there was a significant increase in hepatic cell replication rates. All of these changes persisted from the time they were first observed until the end of the study at 50 weeks. They occurred in a dose-dependent manner and were only observed at doses (500, 1250, 2500 ppm) where liver tumors were increased in the cancer bioassay. There was a clear no-effect level of 200 ppm. The changes seen in this study are consistent with the development of liver cancer in mice and form the basis of the mode of action. When the major metabolites of thiamethoxam, CGA322704, CGA265307, and CGA330050 were tested in dietary feeding studies of up to 20 weeks duration, only metabolite CGA330050 induced the same changes as those seen in the liver in the thiamethoxam feeding study. It was concluded that thiamethoxam is hepatotoxic and hepatocarcinogenic as a result of its metabolism to CGA330050. Metabolite CGA265307 was also shown to be an inhibitor of inducible nitric oxide synthase and to increase the hepatotoxicity of carbon tetrachloride. It is proposed that CGA265307, through its effects on nitric oxide synthase, exacerbates the toxicity of CGA330050 in thiamethoxam treated mice.

Cadmium Absorption in Mice: Effects of Broiling on Bioavailability of Cadmium in Foods of Animal Origin

The absorption and organ distribution of organic Cd from raw and broiled horse kidney was compared to that of CdCl2 at two dose levels (0.05 and 3 mg Cd/kg feed) in a feeding study in mice. The high Cd concentration in the horse kidney (raw 112 mg/kg; broiled 53 mg/kg) made it possible to mix kidney into mouse feed without marked effects on the composition of the feed. The weight of the mice, feed and water consumption, and Cd levels in the feed were determined once a week. After 9 wk of exposure, the liver and kidneys of the mice were sampled and Cd was analyzed. The Cd concentration in horse kidney was halved by broiling, and the content of soluble Cd decreased from 12% in raw kidney to 5% in broiled kidney. The majority of the soluble Cd was associated with proteins with the same molecular weight as metallothionein (MT) in both raw and broiled kidney. Broiling of the kidney had no marked effect on the fractional accumulation of organic Cd in mice. The fractional accumulation of inorganic CdCl2, on the other hand, was significantly higher than that of organic Cd in the low dose groups but not in the high dose groups. The ratio between Cd accumulation in kidney and that in liver was higher in the group receiving raw kidney compared to the ratio in the group receiving CdCl2 at both the high and low exposure levels. This indicates that the raw kidney contained a Cd form that was more preferentially distributed to the kidneys.

CHRONIC TOXICITY AND ONCOGENICITY OF N-METHYLPYRROLIDONE (NMP) IN RATS AND MICE BY DIETARY ADMINISTRATION

A two-year feeding study in rats and an 18-month feeding study in mice were conducted to evaluate the potential chronic toxicity and oncogenicity of NMP in Crl : CD® (SD)BR rats and B6C3F1/CrlBR mice. Groups of 62 male and female rats were administered diets containing 0, 1600, 5000, or 15 000 ppm of NMP for approximately 2 years. Groups of 50 male and female mice were administered diets containing 0, 600, 1200, or 7200 ppm NMP for approximately 18 months. In vivo parameters were evaluated weekly during the first 3 months of the study, and every other week or monthly during the remainder of the study. For rats, an ophthalmoscopic examination was conducted prior to study start and near the end of the study. Periodically, blood samples were collected from rats and mice for determination of leukocyte differential counts, and from mice for red blood cell morphology. After approximately 2 years of dietary administration in rats and 18 months in mice, all surviving animals were sacrificed. Selected tissues were processed for morphological evaluation. Over the course of the two-year study in rats, test substance-related decrements in body weight and weight gain occurred in 15 000 ppm males and females, which correlated with decreased food consumption and food efficiency. A toxicologically significant, test substance-related increase in the incidence of severe chronic progressive nephropathy occurred in 15 000 ppm males. Several morphological changes noted grossly and/or microscopically were secondary to the increased severity of chronic progressive nephropathy. NMP was not oncogenic in male or female rats at dietary concen trations of 15000 ppm and below. A test substance-related decrease in the percentage of 15 000 ppm males surviving to the end of the two-year study compared to the control group resulted from the higher incidence of severe chronic progressive nephropathy. However, a sufficient population of 15000 ppm rats were at risk for potential oncogenicity, so the lower survival did not impair the ability to detect an oncogenic response in this study. There were no adverse, test substance-related effects on the incidences of clinical observations, ophthalmic observations, or differential leukocyte counts in males or females, or on survival of females at any dietary concentration. Male and female mice administered dietary concentrations of 7200 ppm had significantly increased liver weight, significantly increased incidence of hepatocellular adenoma, and significantly increased foci of cellular alteration in the liver. At 7200 ppm, male mice also had an increased incidence of hepatocellular carcinoma while the increased incidence of hepatocellular carcinoma in female mice fell within the historical control range. In addition, the incidence of hepatocellular hypertrophy was increased in 7200 ppm males. Liver weight and hepatocellular hypertrophy were also increased in 1200 ppm males. There were no adverse, test substance-related effects on the incidences of clinical observations, food consumption, body weight, differential leukocyte counts, red blood cell morphology, or survival in either males or females at any dietary concentration. Under the conditions of the study, the no-observed-effect level for NMP was 5000 ppm for male and female rats, 600 ppm for male mice, and 1200 ppm for female mice.

Final Report on the Safety Assessment of Isobutylparaben and Isopropylparaben

Isobutylparaben and Isopropylparaben are esters with a phenol ring structure intended for use as preservatives in cosmetics. In 1993, only Isobutylparaben was reported to be in use. When administered to rabbits via a stomach tube, Isobutylparaben was metabolized primarily to p-hydroxybenzoic acid; &lt;1% was recovered unchanged. Spleen, thymus, hepatic parenchyma, and lymph node atrophy was observed in short-term feeding studies in mice with Isobutylparaben at a concentration of 1.25%. All mice died at concentrations of 5 and 10%. Isobutylparaben at a concentration of 0.6% produced no adverse effects. Mutagenesis was not observed with either ingredient in Ames tests, but both positive and negative results were seen in chromosome abberation assays of Chinese hamster ovary cells in culture. In chronic mouse-feeding studies, 0.15, 0.3, and 0.6% Isobutylparaben caused no increase in neoplasias or decrease in time to neoplasm development. Data on Methylparaben, Ethylparaben, Propylparaben, and Butylparaben considered relevant to the assessment of Isobutylparaben and Isopropylparaben were summarized. Subchronic and chronic studies indicate that these other parabens are practically nontoxic, nonmutagenic, and noncarcinogenic. They also do not irritate or sensitize normal skin. Tests of patients with damaged skin, however, do show that a few of these individuals can be sensitized. These data support the recommendation that all six of these parabens should not be used on damaged skin. Given the absence of any adverse effects, including irritation and sensitization, in persons with normal skin, the conclusion that Methylparaben, Ethylparaben, Propylparaben, and Butylparaben are safe as cosmetic ingredients was reaffirmed. By extension, because of their similarities, the conclusion was reached that Isobutylparaben and Isopropylparaben are safe as cosmetic ingredients in the present practices of use.