Repeating of emotional stress prevents development of melanocortin obesity and type 2 diabetes in the mice with the Agouti yellow mutation

Brain melanocortin system (MC-system) participates in regulation of energy homeostasis. Dominant mutation yellow of the Agouti gene leads to the hyperphagia, obesity and type 2 diabetes. Stress is known to inhibit food intake and body weight. The aim of the work was to study effects of repeating emotional stress on food intake and lipid-carbohydrate metabolism in Ay-mice. Male mice of C57B1/6J strain predisposed to the obesity (Ay/a-genotype) and normal (a/a-genotype) were used. In control group food intake, body weight and blood levels of insulin and leptin were increased in Ay/a-mice as compared to a/a-mice. Repeating emotional stress (30 min restraint 3 times a week for 5 weeks) did not alter food intake and indices of lipid-carbohydrate metabolism in a/a-mice and decreased food intake, body weight and blood levels of insulin and leptin in Ay/a-mice. Insulin and leptin blood levels were the same in Ay/a- and a/a-mice on 5 week of treatment. The stress increased basal and stress-induced concentrations of corticosterone to an equal degree in Ay/a- and a/a-mice. Thus, light repeating emotional stress hampered development of obesity and 2 type diabetes in the mice with the Agouti yellow mutation.     

Steroidogenesis in adrenal glands of young and adult female mice with hyperexpression of the Aguoti gene

Dominant mutation Agouti yellow (AY) leads to ectopic overexpression of the Agouti gene and yellow coat color in mice. Furthermore, the mutation Ay increased adrenal response to emotional stress. The study assessed whether pleiotropic effect of the mutation Ay on adrenals function was dependent on sex and age. 3- and 15-week old female C57B1/6J mice of two agouti-genotypes: Ay/a (ectopic Agouti-gene overexpression) and a/a (absence of Agouti-protein), were investigated. Cyclic AMP level (adenylate cyclase activity) and corticosterone production in adrenal isolated cells stimulated by ACTH and dibutyrul cAMP (db-cAMP) were measured. ACTH increased cAMP accumulation to the same extent in Ay/a- and a/a-mouse adrenal cells of both ages. The dibutyrul cAMP-induced corticosterone production was higher in Ay/a than in a/a-mouse adrenal cells of both ages. The ACTH-induced corticosterone production in 3-week- old Ay/a-m/CQ was lower and in 15-week old Ay/a-mice was higher than in a/a-mice of the respective ages. The ACTH- and db-cAMP-induced steroidogenesis was not changed in Ay/a-mice and decreased in a/a-mice with age. Thus, in females as well as in males, the mutation Agouti yellow did not affect adenylate cyclase activity, increased db-cAMP-induced corticosterone production and disturbed development of adrenal cortex.     

Effect of estradiol on food intake, glucose and fat metabolism in mice C57BL/6J with mutation yellow at the agouti locus

Mutation yellow at the agouti locus in mice (A(y)/a-mice) causes the increase of food intake and development of obesity and type 2 diabetes. In A(y)/a-females the disturbances of glucose and fat metabolisms occur after puberty. We have assumed that the mutation yellow violates the regulatory effect of estradiol on glucose and fat metabolism in mice. We investigated the effects of ovariectomy and estradiol treatment on body weight, food intake, glucose tolerance, plasma levels of glucose, insulin and etherified fatty acids in A(y)/a-females. C57Bl/6J females, not carrying yellow mutation at the agouti locus (a/a-mice), were used as a control. The data suggest that the yellow mutation did not affect estradiol regulation of food intake and glucose blood levels after a night of fasting, but, apparently, prevented estradiol participation in the regulation of glucose and fat metabolisms in the muscle and fat tissues.     

Overproduction of agouti-protein did not affect cAMP level but increased steroidogenesis in adrenal glands under forskolin stimulation

Mutation Agouti yellow (Ay) in mice Ay/a results in overproduction of agouti protein (AP), adult onset of obesity, increased corticosterone responses to restrain stress as compared with a/a mice (absence of AP). The enhanced corticosterone response in restrained Ay/a-mice compared with restrained a/a-mice occurred in result of increased adrenal reactivity to ACTH. The purpose of this work was to investigate the influence of AP overproduction on adenylate cyclase (AC) activity and steroidogenesis in forskolin stimulated adrenal cells. To estimate obesity influence, these parameters were measured in young (3 weeks) and adult (15 weeks) animals. The data obtained demonstrated that AP overproduction and the obesity did not affect the AC activity. However, forskolin stimulated corticosterone production in Ay/a-mice was higher than in a/a-mice (in young--during 0.5 h, in adult--during 3 hrs of incubation). So AP overproduction and obesity affect the corticosterone production. We hypothesize that AP overproduction affects steroidogenesis gene expression: accelerates gene activation in ontogenesis and increases enzyme de novo synthesis during long-term stimulation in adults.     

Morphological characteristics of liver and white adipose tissue during development of melanocortin obesity in C57Bl/6J Agouti yellow mutant mice

The changes of liver and white adipose tissue (WAT) morphology during development of melanocortin obesity in female Agouti yellow (genotype A Y/a) C57Bl/6J mice have been investigated. Mouse strain of a/a genotype was used as a control. Results have been compared with the hormonal and metabolic changes during development of obesity and type 2 diabete in Agouti yellow mice. The tissues obtained from mice of 8-, 11-, 15- and 22-week old have been analyzed. The morphology of liver and WAT of A(Y) and control animals did not differ during 15 weeks of age. 40% of A(Y) mice revealed liver steatosis (fatty liver) at the age of 22 weeks. In addition, elevation in the inflammatory and proliferating processes in the liver and severe inflammation in WAT has been observed in these animals. Since as early as 15 weeks old A(Y) mice demonstrated the appearance of insulin resistance characteristics we conclude that hormonal and metabolic abnormalities could play a role of the primary factor of pathological reorganization of liver and WAT morphology.     

Genetic Studies of the Mouse Mutations Mahogany and Mahoganoid

The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a switch in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for α-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1r(e)) or a gain-of-function Agouti mutation (lethal yellow, A(y)). We found that the Mc1r(e) mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of A(y) on both coat color and obesity. Plasma levels of α-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

Metabolic parameters and functional state of hypothalamic signaling systems in A<Superscript>Y</Superscript>/a mice with genetic predisposition to obesity and the effect of metformin

The metabolic parameters and functional state of hypothalamic systems in mice with the Yellow mutation in the Agouti locus and with obesity of the melanocortin type and the effect of metformin (MF) treatment (9 days, 200 mg/kg/day) were studied. The MF treatment led to decreased body weight and to normalization of glucose tolerance in mice. In the hypothalamus, MF restored the decreased activity of Akt kinase, the main component of leptin pathway, and normalized the increased expression of subtype 1B serotonin receptor. The obtained data suggest the efficiency of MF to treat obesity of the melanocortin type.     

Pituitary-adrenal function in female mice with mutation Agouti yellow (A(y))

Agouti protein is a paracrine signaling factor modulating action of ACTH and alpha-MSH. Dominant mutation Ay causes ectopic, ubiquitous expression of Agouti protein in mice. It was shown that Ay mutation increased stress-induced hypothalamo-adrenal activity in male mice. There is a sex difference in the hypothalamo-pituitary-adrenal axis in rodents. The aim of this study was to test effects of ectopic overexpression of Agouti protein on pituitary-adrenal function in female mice. Female mice of C57Bl/6J strain with Ay mutation (Ay/alpha) and with mutation nonagouti (alpha/alpha; lack of Agouti protein) were used. Ay/alpha-females had an increased blood level of corticosterone and ACTH after 10-minute restriction as compared with alpha/alpha-females. The adrenal threshold sensitivity and reaction to exogenous ACTH in vivo suggests that increased corticosterone reaction to emotional stress is caused by increased pituitary stimulation.     

Function of the hypothalamo-pituitary-adrenal system in mice with ectopic overexpression of agouti protein

Agouti protein (AP) is known to antagonise the effects of melanocortins (ACTH, MSH) on the melanocortin receptors which participate in regulation of central and peripheral HPA links. This study aimed at estimation of effects of dominant mutation Agouti yellow (Ay-ectopic overexpression Agouti protein) on the HPA axis function in mice. Male mice of C57B1/6J strain of Ay/a- and a/a genotypes (control animals, lack of AP), were used. We demonstrated that basal corticosterone level in the Ay mice matched those of control animals. Stress-activated corticosterone level (p < 0.02) and sensitivity of adrenal to low doses ACTH in vitro and in vivo were higher in Ay mice compared with control mice. Dexamethazone-inhibition of stress-reactivity was more intensive in Ay/a than in a/a mice (p < 0.0007).     

A Transgenic Mouse Assay for Agouti Protein Activity

The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.     

Distribution of Mahogany/Attractin mRNA in the rat central nervous system

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.     

Dorsoventral patterning of the mouse coat by Tbx15

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de(H)), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a(t)) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de(H) mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de(H) that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de(H)/de(H) animals. Construction of a targeted allele of Tbx15 confirmed that the de(H) phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.     

不同配方益生菌在高脂饮食诱导小鼠肥胖发生中的差异化作用

【目的】基于肠道微生物与宿主代谢的相互关系,研究不同配方的益生菌对小鼠肥胖的影响。【方法】50只C57BL/6J雄性小鼠随机平均分成10组,分别给予正常饲料、高脂饲料以及高脂饲料加8种不同配方的益生菌产品(50亿CFU/只),所有动物连续喂养9周,每周测量小鼠体重1次。最后一周测定空腹血糖、葡萄糖耐量试验(glucose tolerance test,GTT)、血脂相关指标,称取内脏重量,并留取小鼠盲肠内容物,提取小鼠肠道菌群总DNA,利用16S rDNA测序检测相关细菌含量。【结果】部分益生菌可引起小鼠体重增速加快,而部分益生菌可减缓小鼠肥胖和降低内脏脂肪重量,同时缓解高血脂症。丹尼斯克品牌益生菌配方组小鼠肠道中厚壁菌/拟杆菌比例(F/B)是正常饮食组的22.8倍,Akkermansia muciniphila(Akkermansia)细菌含量几乎为0;而菌拉丁品牌益生菌配方组小鼠F/B比例与正常饲料饮食组类似,Akkermansia含量为0.5%,为正常饮食对照组小鼠的一半左右。【结论】益生菌可影响小鼠体重和代谢,但不同配方的益生菌效果截然相反。特定的益生菌配方对肥胖和高血脂的改善可能是由于其选用的菌株本身的特性以及菌株之间的相互配比能够降低小鼠肠道中F/B比例以及升高Akkermansia的含量所带来的。此研究为进一步开发可改善代谢的益生菌产品提供了参考。     

裂殖壶藻藻油DHA对高脂饮食诱导肥胖小鼠的影响

【目的】肥胖症是一种慢性代谢类疾病,具有较高的发病率和高危后果。研究表明,n-3多不饱和脂肪酸(n-3 Polyunsaturated fatty acids,n-3 PUFAs),特别是二十二碳六烯酸(Docosahexaenoic acid,DHA)对与肥胖症相关疾病有较好的防治效果,对体内脂质代谢有重要的调节作用。探讨裂殖壶藻(Schizochytrium sp.)藻油DHA对高脂饮食诱导肥胖小鼠体重、脂肪组织重量、血脂、肝和脂肪组织病理形态和脂质代谢相关基因表达的影响。【方法】通过高脂饮食建立小鼠肥胖模型,以体重增幅15%为标准分出肥胖小鼠。试验共分五组:(1)低脂对照组;(2)高脂模型组;(3)高脂+低剂量藻油组(50 mg DHA/kg);(4)高脂+中剂量藻油组(100 mg DHA/kg);(5)高脂+高剂量藻油组(200 mg DHA/kg)。其中,藻油处理组灌服相应剂量藻油,低脂对照组和高脂模型组灌胃同等体积玉米油。处理9周后,腹腔麻醉,摘眼球取血并分离血清,测血清中甘油三酯、胆固醇和高密度脂蛋白含量;之后处死小鼠,分离附睾、肾周和肠系膜脂肪组织及肝脏,称湿重;附睾脂肪和肝组织切片进行HE染色,观察病理变化情况;利用RT-PCR检测附睾脂肪组织中激素敏感脂酶(Hormone sensitive lipase,HSL)基因的m RNA表达情况。【结果】藻油处理组小鼠体重没有显著下降,但是腹部脂肪重量显著降低、脂肪细胞体积明显小于高脂模型组;同时血清中甘油三酯、胆固醇含量显著降低,肝组织异位脂肪堆积明显减少;脂肪组织中HSL基因的表达水平显著提高。【结论】裂殖壶藻藻油DHA处理能显著降低高脂饮食导致的小鼠腹部脂肪积累并改善血脂,可能有利于肥胖症的防治。     

Agouti Alleles Influence Thiol Concentrations in Hair Follicles and Extrafollicular Tissues of Mice (Ay/a,AwJ/AwJ,a/a)

Agouti protein (AP) expression in the wild-type agouti mouse (A^wJ/A^wJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (A^y/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of A^y/a, A^wJ/A^wJ, and a/a (black) mice, and 2) adipose and spleen tissues of A^y/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. A^y/a hair follicles and serum showed highest cysteine and lowest GSH levels. A^wJ/A^wJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed A^y/a > a/a (P > 0.01). An A^y-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.     

High Energy Digestion Efficiency and Altered Lipid Metabolism Contribute to Obesity in BFMI Mice

To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual‐energy X‐ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6–10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass–adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia.     

Loss of FXR protects against diet-induced obesity and accelerates liver carcinogenesis in ob/ob mice

Farnesoid X receptor (FXR) is known to play important regulatory roles in bile acid, lipid, and carbohydrate metabolism. Aged (>12 months old) Fxr(-/-) mice also develop spontaneous liver carcinomas. In this report, we used three mouse models to investigate the role of FXR deficiency in obesity. As compared with low-density lipoprotein receptor (Ldlr) knockout (Ldlr(-/-)) mice, the Ldlr(-/-)Fxr(-/-) double-knockout mice were highly resistant to diet-induced obesity, which was associated with increased expression of genes involved in energy metabolism in the skeletal muscle and brown adipose tissue. Such a striking effect of FXR deficiency on obesity on an Ldlr(-/-) background led us to investigate whether FXR deficiency alone is sufficient to affect obesity. As compared with wild-type mice, Fxr(-/-) mice showed resistance to diet-induced weight gain. Interestingly, only female Fxr(-/-) mice showed significant resistance to diet-induced obesity, which was accompanied by increased energy expenditure in these mice. Finally, we determined the effect of FXR deficiency on obesity in a genetically obese and diabetic mouse model. We generated ob(-/-)Fxr(-/-) mice that were deficient in both Leptin and Fxr. On a chow diet, ob(-/-)Fxr(-/-) mice gained less body weight and had reduced body fat mass as compared with ob/ob mice. In addition, we observed liver carcinomas in 43% of young (<11 months old) Ob(-/-)Fxr(-/-) mice. Together these data indicate that loss of FXR prevents diet-induced or genetic obesity and accelerates liver carcinogenesis under diabetic conditions.     

Dose-response effects of ectopic agouti protein on iron overload and age-associated aspects of the Avy/a obese mouse phenome

Isogenic and congenic offspring from matings of inbred black a/a dams by sibling (or non-sibling from another inbred strain) yellow agouti Avy/a sires provide an animal model of obese yellow agouti Avy/a and isogenic lean pseudoagouti Avy/a mice exhibiting two different in vivo concentrations (high, very low) of ectopic agouti protein (ASP) with congenic lean black a/a mice as null controls. This makes it possible to differentiate between the high and very low dose levels of ectopic ASP with respect to interactions with diverse physiological and molecular pathways. Assay of differential responses to 12 or 24 months of carbonyl iron overload assessed the possible suitability of this animal model for the study of hemochromatosis. Agouti A/a B6C3F1 mice were used as non-congenic null controls. The age-related waxing and waning of body weight, food consumption, and caloric efficiency, as well as associated changes in pancreatic islets and islet cells, and formation of liver tumors were assayed. While the hypothesis that these mice might serve as a tool for investigating hemochromatosis was not confirmed, the data did provide evidence that even the very low levels of ASP in pseudoagouti Avy/a mice affect the network of molecular/metabolic/physiological response pathways that comprises the yellow agouti obese phenome. We suggest that the combination of yellow agouti Avy/a, pseudoagouti Avy/a, and black a/a congenic mice provides a practical tool for applying a dose-response systems biology approach to understanding the dysregulatory influence of ectopic ASP on the molecular-physiological matrix of the organism.     

Long-term low-dose mutation studies in human cells: metanil yellow and orange II

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.