Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O.

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59-) or mutant reticulocyte frequencies (RETCD59-) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.

Auramine O, an incense smoke ingredient, promotes lung cancer malignancy.

Burning incense to worship deities is a popular religious ritual in large parts of Asia, and is a popular custom affecting more than 1.5 billion adherents. Due to incomplete combustion, burning incense has been well recognized to generate airborne hazards to human health. However, the correlation between burning incense and lung cancer in epidemiological studies remains controversy. Therefore, we speculated that some unknown materials in incense smoke are involved in the initiation or progression of lung cancer. Based on this hypothesis, we identified a major compound auramine O (AuO) from the water-soluble fraction of incense burned condensate using mass spectrometry. AuO is commonly used in incense manufacture as a colorant. Due to thermostable, AuO released from burned incenses becomes an unexpected air pollutant. AuO is classified as a Group 2B chemical by the International Agency of Research on Cancer (IARC), however, the damage of AuO to the respiratory system remains elusive. Our study revealed that AuO has no apparent effect on malignant transformation; but, it dramatically promotes lung cancer malignancy. AuO accumulates in the nucleus and induces the autophagy activity in lung tumor cells. AuO significantly enhances migration and invasive abilities and the in vitro and in vivo stemness features of lung tumor cells through activating the expression of aldehyde dehydrogenase family 1 member A1 (ALDH1A1), and ALDH1A1 knockdown attenuates AuO-induced autophagy activity and blocks AuO-induced lung tumor malignancy. In conclusion, we found that AuO, an ingredient of incense smoke, significantly increases the metastatic abilities and stemness characters of lung tumor cells through the activation of ALDH1A1, which is known to be associated with poor outcome and progression of lung cancer. For public health, reducing or avoiding the use of AuO in incense is recommended.

Toxicity of imine-iminium dyes and pigments: electron transfer, radicals, oxidative stress and other physiological effects.

Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer-reactive oxygen species-oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer-reactive oxygen species-oxidative stress arising from imine-iminium.

Free radicals generated in yeast by the Salmonella test-negative carcinogens benzene, urethane, thiourea and auramine O.

A large fraction of carcinogens score negative in short-term genotoxicity assays such as the Salmonella reverse mutation (Ames) assay. More information is needed about the mechanism of action of such Salmonella-negative carcinogens. Many Salmonella-negative carcinogens induce deletions due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We have previously shown that the Salmonella-negative carcinogens cadmium, aniline, chloroform and carbon tetrachloride generate free radical species in S. cerevisiae. We have further investigated the possible generation of intracellular free radical species by the diverse Salmonella-negative carcinogens benzene, urethane, thiourea and auramine O. The toxicity and recombinagenicity of thiourea and auramine O was reduced in the presence of the free radical scavenger N-acetyl cysteine. N-acetyl cysteine did not protect against toxicity or recombination induced by the Salmonella-positive carcinogens ethyl methane sulfonate, methyl methane sulfonate or nitroquinoline-N-oxide. A strain deficient in the enzyme superoxide dismutase, which catalyses the dismutation of superoxide anion radical, was hypersensitive to killing by benzene, urethane and thiourea. The sod- strain was only slightly more sensitive to the Salmonella-positive carcinogens. Intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate was increased in yeast cultures exposed to benzene, urethane and auramine O; again, the Salmonella mutagens had no effect on oxidation of the dye. These data show that free radical species are produced in Saccharomyces cerevisiae following exposure to benzene, urethane, thiourea and auramine O, and suggest a possible role for oxidative stress is recombination induced by these carcinogens.

Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat.

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 microM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg-1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.

Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow).

We have recently designed a simple method for applying the alkaline single-cell gel electrophoresis (SCG) assay to mouse organs. With this method, each organ is minced, suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, gently homogenized using a Potter-type homogenizer set in ice, and then centrifuged nuclei are used for the alkaline SCG assay. In the present study, we used the method to assess the genotoxicity of 8 rodent hepatic carcinogens in 5 mouse organs (liver, lung, kidney, spleen, and bone marrow). The carcinogens we studied were p-aminoazobenzene, auramine, 2,4-diaminotoluene, p-dichlorobenzene, ethylene thiourea (ETU), styrene-7,8-oxide, phenobarbital sodium, and benzene-1,2,3,4,5,6-hexachloride (BHC); except for p-aminoazobenzene, they do not induce micronuclei in mouse bone marrow cells. Mice were sacrificed 3 and 24 h after the administration of each carcinogen. p-Aminoazobenzene, ETU, and styrene-7,8-oxide induced alkaline labile DNA lesions in all of the organs studied. Auramine, 2,4-diaminotoluene, p-dichlorobenzene, and phenobarbital sodium also produced lesions, but their effect was greatest in the liver. BHC, which is not genotoxic in in vitro tests, did not show any effects. We suggest that it may be possible to use the alkaline SCG assay to detect in vivo activity of chemicals whose genotoxicity is not expressed in bone marrow cells.

DNA damage induced by auramine O in liver, kidney, and bone marrow of rats and mice, and in a human cell line (alkaline elution assay and SCE induction).

Auramine O has been reported to be carcinogenic in rats and mice. It has been reported as positive in some mutagenicity studies and negative in others. We have found that commercial auramine O is positive in inducing DNA damage in vivo in liver, kidney, and bone marrow cells. DNA damage was also induced after treatment in vitro of a human cell line. Commercial auramine O was also clearly positive for sister chromatid exchange (SCE) induction in vivo in bone marrow cells. Purified auramine was negative in terms of DNA damage and SCE induction. Our commercial auramine O had Michler's ketone as a major contaminant. This compound was capable of inducing both DNA fragmentation and an increase of SCE.

[Reaction of the seedlings of flowering plants to the effects of imino-di-(paradimethylaminophenyl)-methane-aurine-00]. / Reaktsiia propostkov tsvetkovykh rastenii na vozdeistvie imino-di-(paradimetilaminofenil)-metana-auramina-00

The influence of auramine-00 on seedlings of three species of plants - Cucurbita pepo L., Helianthus annuus L., Zea mays L.-was investigated. Auramine was added in agar-agar, on which the seedlings were growing. Two concentrations of auramine were used--0.001%. Besides the delay in growth, also the formation of local overgrowth of cotyledons and hypocotyl of C. pepo, the base of cotyledons of H. annuus and mezocotyl of Z. mays were observed. The histological analysis has shown these swellings to arise on the grounds of two processes: pathological proliferation and hypertrophy.