All India survey for analyses of colors in sweets and savories: exposure risk in Indian population.

In the present study, an attempt has been made to understand the exposure assessment of food colors through 2 major groups, sweets and savories, at a national level so as to evolve a scientific yardstick to fix levels of colors in commodities based on technological and safety requirement. A vast majority of colored food commodities (83.6%) were found to employ permitted colors and confirmed a marked decline in the trend of use of nonpermitted colors (NPCs). Of the 4 zones of India, East zone showed the maximum adulteration (80.3%) both by exceeding the prescribed limits of permitted colors (72.3%) and the use of NPCs (28.7%). Tartrazine was the most popular color among the permitted list, which ranged from 12.5 to 1091 mg/kg. Rhodamine B was the most prevalent dye in the NPCs group. On the basis of average consumption of food commodities and average levels of detected colors, the intake of Sunset Yellow FCF saturates the acceptable daily intake limit to a maximum of 47.8% in children, which is a cause of concern. The uniform maximum permissible limit of synthetic colors at 100 mg/kg under the Indian rules thus needs to be reviewed and should rather be governed by the technological necessity and the consumption profiles of food commodities so that the vulnerable population should not unnecessary be exposed to excessive amounts of synthetic colors to pose health risks.

A simple method for simultaneous determination of basic dyes encountered in food preparations by reversed-phase HPLC.

The present method utilizes a simple pretreatment step, cleanup on polyamide SPE cartridges, and HPLC resolution on reversed-phase C18 for the detection of the three basic nonpermitted dyes encountered in food matrixes. Polyamide cartridges were chosen because both acidic and basic dyes can be cleaned up due to their amphoteric nature. Analysis was performed on a reversed-phase C18 micro-Bondapak column using the isocratic mixture of acetonitrile-sodium acetate with a flow rate of 1.5 mL/min and a programmable lambda(max) specific visible detection to monitor colors, achieving higher sensitivity and expanded scope to test multicolor blends. All the colors showed linearity with the regression coefficient, from 0.9983 to 0.9995. The LOD and LOQ ranged between 0.107 and 0.754 mg/L and 0.371 and 2.27 mg/L or mg/kg, respectively. The intraday and interday precision gave good RSDs, and percentage recoveries in different food matrixes ranged from 75 to 96.5%. The study demonstrates that the use of a combination of a simple SPE cleanup and HPLC resolution with UV-Vis end point detection was successful in screening the presence of these three basic nonpermitted dyes individually or in blend, in a variety of food matrixes.

Simultaneous determination of eight synthetic permitted and five commonly encountered nonpermitted food colors in various food matrixes by high-performance liquid chromatography.

A simple and sensitive HPLC method has been developed for the simultaneous determination of eight permitted food colors and five commonly encountered nonpermitted colors in various food commodities, including sugar-, fat-, and starch-based food matrixes. The method uses a specific food category-based cleanup/treatment procedure before color extraction to avoid the interference of food matrixes, and to obtain the optimal color extraction. Analysis was performed on a reversed-phase C18 micro-Bondapak column with ammonium acetate and acetonitrile gradient elution as the mobile phase; a programmable lamda max-specific visible detection was used to monitor colors to obtain the higher sensitivity and expanded scope needed for multicolor blends having diverse absorption maxima. All colors showed good linearity, with regression coefficients of 0.9974-0.9999. The LOD and LOQ values ranged from 0.01 to 0.12 mg/L, and from 0.04 to 0.83 mg/L or mg/kg, respectively. The intraday and interday precision tests produced good RSD values, and the recoveries from different food matrixes ranged from 82 to 104%. The method offers high sensitivity for analysis of a wide variety of food matrixes containing a broad scope of multicolor blends. Two nonpermitted colors, orange II and metanil yellow, were found. Also, a number of samples contained permitted colors at levels two- to seven-fold higher than those prescribed.

Potentiation of neurotoxicity of Lathyrus sativus by manganese: alterations in blood-brain barrier permeability.

Environmental factors have been speculated to play an important role in potentiating the neurotoxicity of Lathyrus sativus (LS). Hence, blood-brain barrier permeability and neurotoxicity studies were carried out in manganese- and LS-exposed animals. Dietary feeding of LS (80%) plus Mn (0.4 mg/100 g diet) for 90 days to guinea pigs showed significant (p < 0.05) decrease in brain nucleotidase and ATPase activities when compared to control or LS alone treated groups. Combined treatment of LS and Mn showed a significant (p < 0.05) decrease in neuronal aryl hydrocarbon hydroxylase (36-40%), ethoxyresorufin-O-deethylase (40-45%), glutathione-S-transferase (27-31%), and quinone reductase (24-25%) activities when compared to control and LS alone treated animals. Lipid peroxidation, a marker for membrane damage, was found to be relatively more enhanced (58-141%) along with significant (p < 0.05) depletion of GSH levels in LS+Mn-treated animals when compared to control, Mn alone, and LS alone treated groups. The neuronal catalase activity of lathyrus plus Mn-treated animals showed a pronounced decrease (37-49%) when compared to control, Mn, and lathyrus alone treated groups. On the contrary, glutathione peroxidase in brain of Mn and lathyrus alone treated animals indicated a respective increase (p < 0.05) of 18% and 20%, while the combined effect of lathyrus plus Mn exhibited an increase of almost 50% when compared to control guinea pigs. Single parenteral administration of Mn (15 mg/kg b.wt) to guinea pigs followed by single oral intubation of beta-N-oxalyl-L-alpha, beta-diamino propionic acid (ODAP, 75 mg/guinea pig) resulted in a significant increase (143%) in neuronal ODAP content. ODAP (50 mg/kg,iv) treatment to mice pretreated with MnCl2 (10 mg/kg b.wt for 3 days or 40 mg/kg b.wt for 1 day), caused an enhancement in blood-brain barrier (BBB) permeability (129-196%), while ODAP and Mn alone showed relatively less enhancement (66-87%). The lumbar region of LS+Mn showed a number of vacuolated areas of variegated size and chromatolytic neurons, along with a few degenerated neurons. These results suggest that Mn may potentiate the neurotoxicity of lathyrus/ODAP by altering the BBB permeability.

Alterations in redox potential of glutathione/glutathione disulfide and cysteine/cysteine disulfide couples in plasma of dropsy patients with argemone oil poisoning.

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. In the present study, we determined the relationship between redox potentials (E(h)) of glutathione/glutathione disulfide (GSH/GSSG), cysteine/cysteine disulfide (Cys/CySS) couples and non-enzymatic antioxidants such as alpha-tocopherol and ascorbic acid status in plasma of dropsy patients (n=14) from an outbreak of argemone oil poisoning in Lucknow (March, 2005), India. Depleted GSH (55%) and concomitant enhancement (163%) of plasma GSSG content was observed in patients (P<0.05). Furthermore, lower content of Cys (42%) and CySS (25%) was noticed in patients (P<0.05) when compared to control subjects. Eh GSH and Eh Cys values were shifted by +46 mV and +12 mV towards more oxidizing environment in patients (P<0.05). In addition, alpha-tocopherol and ascorbic acid contents were found to be depleted significantly (P<0.05) in plasma of patients (59-58%). The alterations in redox potentials and antioxidants in plasma, which are synthesized in liver, may be responsible for histopathological changes in hepatic tissue of patients showing swelling of hepatocytes, fluid accumulation in spaces of Desci along with mild kupfur cell hyperplasia. Over all the present study shows that redox state of GSH/GSSG and Cys/CySS pools become oxidized which inturn causes depletion of alpha-tocopherol and ascorbic acid, thus providing a strategy to distinguish pro-oxidant and antioxidant events in patients.

Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning.

Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42-52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120-144%) in patients and AO treated animals (112-137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in alpha-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59-70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of alpha-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy.

A simple 2-directional high-performance thin-layer chromatographic method for the simultaneous determination of curcumin, metanil yellow, and sudan dyes in turmeric, chili, and curry powders.

A method using simple extraction and 2-directional high-performance thin-layer chromatography (HPTLC) was developed for the simultaneous determination of curcumin, metanil yellow, and sudan dyes in turmeric, chili, and various mixed curry powder formulations. The method offers resolution (Rf) of turmeric pigments, namely, curcumin (0.77), demethoxycurcumin (0.69), bis(demethoxy)curcumin (0.61), and the synthetic dye metanil yellow (0.05) by the first-directional mobile phase, chloroform-methanol (9 + 1, v/v). The resolution (Rf) of sudan I (0.30) and sudan IV (0.23) was achieved by the second-directional mobile phase, toluene-hexane-acetic acid (50 + 50 + 1, v/v/v). Natural pigments of both turmeric and chili showed no interference in the detection and quantification of synthetic colors. The limit of detection and limit of quantification values for curcumin, metanil yellow, sudan I, and sudan IV were 17.39, 42.90, 15.45, and 7.01 and 52.71,130.0, 46.80, and 21.24 ng/spot, respectively. Analysis of a few market samples showed the presence of metanil yellow (1.5-4.6 mg/g), sudan I (4.8-12.1 mg/g), and sudan IV (0.9-2.0 mg/g) in loose turmeric and chili samples, whereas the curcumin content in turmeric and mixed curry powder samples ranged from 6.5 to 36.4 and from 0.3 to 1.9 mg/g, respectively. The method is relatively simple, offers reasonable sensitivity, and can be used to screen a large number of samples.

Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice.

A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-methylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. The skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the following mechanisms: (i) by acting as an antioxidant; (ii) by modulating phase I and II enzymes; (iii) by exhibiting antiproliferative activity.

Usage of saccharin in food products and its intake by the population of Lucknow, India.

A survey of the usage patterns of the artificial sweetener, saccharin, in edible products and a study of its intake pattern in different population groups has been carried out. Of the different edible commodities, ice candy (87 samples) and crushed ice (14 samples), commonly consumed by children, and pan masala (16 samples) and pan flavourings (10 samples), consumed by the habitual population, were collected from different areas of Lucknow, India. Saccharin was extracted from the samples according to an AOAC method and analysed by HPLC. The consumption pattern of ice candy and crushed ice was determined for 6-20 year olds from a household dietary survey using the food frequency recall method (414 families having 1039 subjects). The consumption of pan masala and pan was assessed by a survey of habitual adult consumers comprising 782 and 1141 subjects, respectively. The average and maximum amounts of saccharin in pan masala samples were 12 750 and 24 300 mg kg-1, respectively, which are 1.6- and 3-fold higher than the maximum permitted levels allowed under Prevention of Food Adulteration (PFA) Act of India. In pan flavourings, the average and maximum amount of saccharin was 12.2 and 20.1%, i.e. 1.52- and 2.5-fold higher than the permissible limits of the PFA Act. The samples of ice candy and crushed ice showed average and maximum levels of 200 and 700 mg kg-1 and 280 and 460 mg kg-1, respectively. The average intake of saccharin through ice candy and crushed ice was less than 21% of the acceptable daily intake (ADI) (5 mg kg-1 body weight (bw) day-1). However, the maximum intake of saccharin, especially in the 6-10-year age group, contributed 57 and 68% of the ADI through ice candy and crushed ice, respectively. Maximum consumption of saccharin in all the age groups, if consuming both ice candy and crushed ice, results in exceeding the ADI by 54% for subjects in the 6-10-year age group. Hence, the 6-10-year age group population may be at risk of exceeding the ADI for saccharin. The average and maximum theoretical daily intake of saccharin through pan masala alone was 1.84 and 13.33 mg kg-1 bw day-1, contributing 37 and 267% of the ADI, whereas the estimated (maximum) daily intake was 810% of the ADI. The estimated maximum daily intake (EDI) of saccharin through pan was 6.87 mg kg-1 bw day-1, which was 137% of the ADI. Thus, individuals in the maximum consumption group for pan masala or pan may be susceptible to toxic effects of saccharin, including bladder distention, elevated urine osmolality and bladder cancer.

Safety evaluation studies on argemone oil through dietary exposure for 90days in rats.

Epidemic dropsy is a disease caused by the consumption of mustard oil contaminated with argemone oil (AO). During 1998 dropsy in New Delhi, which is so far the largest with more than 3000 victims and over 60 deaths, it was enquired at various scientific and regulatory meetings about the maximum tolerated dose of AO. Hence, the present study was aimed to investigate the safety levels of AO in rats. Animals were given AO in diet at a dose of 0.001%, 0.01%, 0.1%, 0.5% and 1% daily for 90 days and the two control groups received the standard diet with and without 1% mustard oil. A decrease in body weight gain (28-31%) was observed in 0.5% and 1% AO groups; while significant increases in relative lungs and liver weight was noticed in respective doses of 0.01% and 0.1% AO groups as well as in higher dosage animals. Reduction in RBC count and hemoglobin content (p<0.05) was noticed in 0.01% and 0.1% AO exposed animals. This effect was more pronounced in higher AO doses. Serum marker enzymes including alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) were found to be significantly elevated in 0.01-1% AO groups. Further, a decrease in albumin/globulin ratio (42-78%) was observed in the serum of 0.01% to higher AO dose groups. The levels of serum triglycerides and VLDL cholesterol were found to be enhanced (p<0.05) in AO treated (0.01-1.0%) animals. Histopathological changes in lung were observed at 0.01% dose of AO while liver, kidney and heart produced changes at 0.1% AO and above doses. None of the parameters were found to be affected in 0.001% AO treated animals. These results suggest that the no observed adverse effect level (NOAEL) dose of AO is 0.001% in rats and considering a factor of 100 for humans for highly toxic compound, the safe limit of 0.00001% (100 ppb or 100 ng AO/g oil) AO can be implicated which shall contain only 0.55% of sanguinarine equivalent to 0.6 ng sanguinarine per gram oil. However, the minimum detectable limit of AO is 5 ppm (equivalent to 5 microg sanguinarine per gram oil) with the present existing HPLC method, thereby suggesting that mustard oil should be absolutely free from AO contamination.

Protective effect of bioantioxidants on argemone oil/sanguinarine alkaloid induced genotoxicity in mice.

Our prior studies have shown that argemone oil (AO) and its alkaloid sanguinarine causes DNA damage in mice and Epidemic Dropsy patients. Since some of the bioantioxidants including riboflavin and alpha-tocopherol offered protection to Epidemic Dropsy patients, a combination of riboflavin and alpha-tocopherol was evaluated on AO and sanguinarine induced genotoxicity using alkaline comet assay. Single administration of combination of riboflavin (50mg/kg) and alpha-tocopherol (150mg/kg) to mice, 24h prior to or immediately after AO (2.0ml/kg) exposure showed significant decrease in tail moment (70-72%), tail length (37-44%), and tail DNA (49-53%) in bone marrow cells. Single or multiple doses of antioxidants given after 24h of AO exposure resulted in substantial (P<0.05) decrease in all the parameters of comet assay in bone marrow cells. Single dose of antioxidants given either 24h prior to or immediately after sanguinarine (21.6mg/kg) exposure caused significant decrease in tail moment (56-62%), tail length (69%) and tail DNA (34-42%) in bone marrow cells of mice. Single or multiple doses of antioxidants given after 24h of sanguinarine treated resulted in decrease in tail moment (50-71%), tail length (54-63%) and tail DNA (29-43%) in bone marrow cells. Similar protective response of combination of antioxidants was observed in blood cells of mice treated either with AO or sanguinarine alkaloid. Further, the frequency of bone marrow and blood cells in Olive tail moment category of 8 and onwards were found to be substantially reduced in antioxidants treated animals as compared to respective AO or sanguinarine exposed mice. Based on these results, it can be suggested that a combination of riboflavin and alpha-tocopherol provides protection against AO and sanguinarine induced genotoxicity.

Skin tumorigenic potential of aflatoxin B1 in mice.

Aflatoxin B1 (AFB1) has been classified as a category I human carcinogen, which is responsible for a high incidence of hepatocellular carcinoma. Since exposure to AFB1 can occur through skin contact in addition to ingestion and inhalation, the carcinogenic potential of topically applied AFB1 on mouse skin was investigated. Our results show that single topical application of AFB1 (80 nmol) as a tumor initiator, followed by twice weekly application of 12-tetradecanoyl phorbol myristate acetate (TPA, 4 nmol), resulted in tumor formation after 13 weeks. However, no tumorigenic potential was observed when AFB1 (16 nmol) was used either as a complete carcinogen or as a tumor promoter (4 nmol). Histological analysis of skin showed squamous cell carcinoma in the AFB1/TPA treated group. The application of AFB1 as a complete carcinogen, an initiator or a promoter after 24 weeks demonstrated widespread degenerative and necrotic changes in hepatic tissue as well, suggesting liver to be the target organ following percutaneous absorption. Additionally, twice weekly topical application of AFB1 caused significant induction of cutaneous CYP IA monoxygenases without any effect on hepatic levels while glutathione-S-transferase activity was induced more in the liver than skin. The topical application of AFB1 also resulted in increased hepatic and cutaneous lipid peroxidation with concomitant depletion of glutathione content. It is likely that due to higher induction of hepatic GST activity, products of lipid peroxidation may be detoxified and therefore unable to cause DNA damage making mice resistant to hepatic tumor formation. The overall results indicate a tumor initiating potential of AFB1 in mice and suggest that continued dermal exposure of AFB1, even at low doses, might lead to degenerative changes in hepatocytes.

Correlation of DNA damage in epidemic dropsy patients to carcinogenic potential of argemone oil and isolated sanguinarine alkaloid in mice.

In recent times, a higher incidence of gall bladder carcinoma in the Indo-Gangetic basin has been linked with the consumption of contaminated mustard oil. Consumption of mustard oil contaminated with argemone oil (AO) is well known to cause clinical manifestation referred to as "epidemic dropsy." Because sanguinarine, an active alkaloid of AO, has been shown to intercalate DNA, a possible correlation of DNA damage in epidemic dropsy patients to tumorigenic potential of AO and isolated sanguinarine alkaloid in mice was investigated in the present study. Single topical application of AO (0.15-0.3 ml) or isolated sanguinarine (4.5-18 micromol) followed by twice-weekly application of tetradecanoylphorbolmyristate acetate (TPA) for 25 weeks resulted in formation of tumors. Histopathologically these tumors were of squamous cell carcinoma type and similar to those found in the positive control group using dimethylbenzanthracene (DMBA)/TPA. The activities of cutaneous gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase P (GST-P), marker enzymes of tumorigenesis, were found to exhibit higher expression in AO or isolated sanguinarine/TPA treated groups when compared to control. The higher expression of p53 and p21/WAF1 in skin after single topical application of AO or isolated sanguinarine further confirms the tumorigenic response. Single topical application of AO or isolated sanguinarine alkaloid to mice showed significant DNA damage in terms of Olive tail moment (89-129%), tail length (54%) and tail DNA (153-205%) using Comet assay in skin cells. Further, the extent of DNA damage in blood cells of epidemic dropsy patients in alkaline Comet assay was found to be significantly higher as compared to normal population, indicating the genotoxic response of AO exposure. Although the genotoxic lesions may be repaired to some extent on withdrawal of consumption of AO contaminated mustard oil and the residual genotoxic effects caused by AO may not be expressed as signs of carcinogenesis. Environmental factors or hormonal changes during aging process may lead to stimulate/promote the genetically altered latent cells to form neoplastic lesions and can act as one of the etiological factors responsible for higher incidence of gall bladder carcinoma in the population of Indo-Gangetic basin.

In vivo DNA damaging potential of sanguinarine alkaloid, isolated from argemone oil, using alkaline Comet assay in mice.

Consumption of mustard oil contaminated with argemone oil is well known to cause clinical manifestation referred to as "Epidemic Dropsy". Our prior studies have shown that argemone oil produces genotoxic effects in mice [Ansari, K.M., et al., 2004. Int. J. Cancer 112, 890]. Since, sanguinarine alkaloid is the major component of argemone oil, the in vivo DNA damaging potential of the isolated alkaloid was investigated in blood and bone marrow cells of mice using alkaline Comet assay. Swiss albino male mice were given single intraperitoneal administration of 1.35, 2.70, 5.40, 10.80 and 21.60 mg sanguinarine alkaloid/kg b wt., while controls were treated with saline in the same manner. The results revealed a dose dependent increase in DNA damage in blood and bone marrow cells following 24 h treatment of sanguinarine alkaloid. All the three parameters of Comet assay including olive tail moment (OTM), tail length and tail DNA showed significant (p<0.05) increases in blood and bone marrow cells at respective doses of 10.80 and 5.40 mg alkaloid/kg b wt. However, some of the parameters were significantly increased even at lower doses of sanguinarine alkaloid (2.70 mg/kg b wt.). The frequency of cells exhibiting greater DNA damage were found to be increased by sanguinarine alkaloid in a concentration dependent manner. These results indicate that single exposure of sanguinarine alkaloid causes DNA damage in blood and bone marrow cells of mice, which could be responsible for the genotoxicity of argemone oil.

Mechanism to study 1:1 stoichiometry of NADPH and alkoxyphenoxazones metabolism spectrophotometrically in subcellular biological preparations.

Our prior studies have shown that pentoxyresorufin-O-dealkylation (PROD) can be measured spectrophotometrically with simultaneous monitoring of stoichiometry of NADPH/substrate and NADP/product as 10:1:10:1 [Rastogi et al. FEBS Letters 512 (2002) 121-124]. In the present investigation, mechanism of action of other enzymes in modulating the stoichiometry of alkoxyphenoxazones metabolism to 1:1 for electron donor/substrate and oxidized electron donor/product in the same incubation mixture was studied. The spectrophotometric analysis reveals 10:1 ratio between NADPH and pentoxyresorufin (PRF)-ethoxyresorufin (ERF) in microsomal system. The high ratio of electron donor to substrate is due to the presence of the other forms of P-450, which may participate in endogenous metabolism of compounds, thereby reducing the ratio to 4:1 and 7:1 for NADPH/PRF-ERF. Incubation of dicumarol in the microsomal PROD or ethoxyresorufin-O-dealkylase (EROD) assay led to significant decrease in the consumption of NADPH with a ratio of 4:1 and 7:1 for NADPH/PRF-ERF which is due to inhibition of NADPH cytochrome c (P-450) reductase. In post mitochondrial fraction (S-9), the ratio of 11:1 and 15:1 is seen for NADPH/PRF-ERF. The addition of dicumarol in S-9 fraction showed enhanced rate of alkoxyphenoxazone utilization, suggesting the possibility of reduced resorufin product as a feedback inhibitor. Equating the ratio of NADPH/substrate(s) derived after endogenous utilization of NADPH with the ratio after accounting for NADPH consumption following dicumarol addition in either S-9 or microsomal fraction, a 1:1 mol of NADPH/substrate(s) and oxidized electron donor/product is obtained. The results further suggest that cytosolic fraction may interfere in monitoring the formation of resorufin during dealkylation of alkoxyphenoxazones making dicumarol a mandatory cofactor.

Unequivocal evidence of genotoxic potential of argemone oil in mice.

Consumption of mustard oil adulterated with argemone oil leads to a clinical condition, commonly referred to as "Epidemic Dropsy." Since in vitro studies have shown that sanguinarine, an active benzophenanthridine alkaloid of argemone oil, intercalates DNA molecule, the in vivo clastogenic and DNA damaging potential of argemone oil was investigated in mice. Swiss albino mice were intraperitoneally administered 0.5, 1.0, 2.0 and 4.0 ml/kg body wt. of argemone oil to analyze chromosome aberrations and micronucleus test, while 0.25, 0.5, 1.0 and 2.0 ml/kg body wt. were given for alkaline comet assay. The frequencies of chromosomal aberrations and micronucleated erythrocytes formation in mouse bone marrow cells increased in a dose-dependent manner following argemone oil treatment. However, significant induction in chromosomal aberrations (83%) and micronucleated erythrocytes formation (261%) were observed at a minimum dose of 1.0 ml/kg. The results of comet assay revealed DNA damage in blood, bone marrow and liver cells following argemone oil treatment. Olive tail moment (OTM) and tail DNA showed significant increase in bone marrow (35-44%) and blood cells (25-40%) even at a dose of 0.25 ml/kg body wt. of argemone oil. In liver cells, OTM was significantly increased (20%) at a dose of 0.25 ml/kg, while all the comet parameters including OTM, tail length and tail DNA showed significant increase (31-101%) at a dose of 0.5 ml/kg. These results clearly suggest that single exposure of argemone oil even at low doses produces genotoxic effects in mice.

A novel method for the determination of synthetic colors in ice cream samples.

A simple method has been developed for the extraction, separation, and determination of synthetic colors in ice cream samples. The process involves the breakdown of emulsion by neutral detergents (Triton X-100 and Tween 20) followed by extraction with petroleum ether for removal of fat. The aqueous colored solution obtained is treated with 5% acetic acid, and the uptake of color is carried out by a wool-dyeing technique. The color is eluted from the wool with 5% ammonia solution, the solution is evaporated to dryness, and the residue is dissolved in 60% ethanol for paper chromatography using trisodium citrate-ammonia-water (2 + 5 + 95, w/v/v) as the mobile phase. The colored spots from the paper chromatogram are cut and eluted with 60% ethanol, and the absorbance is measured at the respective lambda maximum corresponding to the Rf value of the appropriate standard. The recoveries of 6 colors, including sunset yellow FCF (SSYFCF), tartrazine, carmoisine, ponceau 4R, brilliant blue FCF (BBFCF), and fast green FCF from spiked samples with either detergent were found to be >90%. However, recoveries of erythrosine were 21 and 65% with Triton X-100 and Tween 20, respectively. Indigo carmine could not be recovered at all because of its fugitive property in 5% ammonia solution, which is used to strip the color from the wool. The sensitivity of the method with the use of Tween 20 is 1 ppm (1 microg/g) for the colors in spiked ice cream samples. With this method, we analyzed samples of 20 branded colored ice cream. The results showed the presence of tartrazine (8.4-43.3 ppm), SSYFCF (23.5-117.6 ppm), carmoisine (traces-53.2 ppm), erythrosine (3.5 ppm), and BBFCF (4.1 ppm) in the ice cream samples. Apart from 2 samples of tuttifruity, all of the ice cream samples showed the presence of permitted synthetic colors below the permissible level of 100 ppm established by the Prevention of Food Adulteration Act of India.

Enhancement of urinary elimination of 3-bromobenzanthrone metabolites by oral supplementation of ascorbic acid in guinea pigs.

OBJECTIVE: 3-Bromobenzanthrone (3-BBA), an anthraquinone intermediate dye, is extensively used in textile industry. Since, our prior studies have shown that 3-BBA caused significant depletion of ascorbic acid (AsA) levels, the effect of exogenous supplementation of AsA on the urinary elimination of 3-BBA metabolites was investigated. METHOD: Guinea pigs were treated with single oral dose of 3-BBA (50 mg/kg b. wt.) in groundnut oil while another group was treated with single oral dose of 3-BBA (50 mg/kg b. wt.) along with 3 day prior and post oral supplementation of AsA. Control groups were either treated with groundnut oil or AsA alone. Urine from individual animals was collected, extracted and analysed on HPTLC. RESULTS: The highest elimination of 3-BBA (75 microg) was found to be in 0-24 h urine fraction which decreased to 18 microg and 5 microg in the two subsequent 24 hourly fractions of urine. Exogenous supplementation of AsA increased the total urinary elimination of 3-BBA by almost 77%. A total of 10 fluorescent metabolites excluding the parent compound were eliminated in the urine of guinea pigs treated with 3-BBA. Densitometric scanning of chromatogram showed different peaks at Rf 0.18, 0.22, 0.27, 0.34, 0.40, 0.48, 0.56, 0.66, 0.72, 0.80, and 0.95 which were eliminated and marked as urinary metabolite 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively. AsA not only significantly enhanced the elimination of 3-BBA metabolites but also modified the pattern of metabolites drastically in 0-6 h, 6-24 h and 24-48 h urine fractions. CONCLUSION: These results indicate that AsA may be useful in protecting the toxicity of 3-BBA by fascilitating the urinary metabolite(s) excretion of 3-BBA.

Bio-elimination of conjugated metabolites of 3-bromobenzanthrone in urine of rats and Guinea pigs.

The profile of urinary metabolites of 3-bromobenzanthrone (3-BBA), an extensively used anthraquinone dye intermediate, in rats and guinea pigs was investigated using HPTLC system. A total of 10 fluorescent metabolites were detected in the urine of guinea pigs as compared to 8 in rats including the parent compound 3-BBA. The elimination of metabolites increased in a dose dependent manner in rats. The Rf values of metabolites in rats were 0.14, 0.29, 0.42, 0.52, 0.58, 0.64, 0.77 and 0.91 while that in guinea pigs were 0.23, 0.26, 0.34, 0.37, 0.44, 0.54, 0.56, 0.68, 0.76 and 0.95. The urine of 3-BBA (50 mg/kg b.wt) treated guinea pigs when digested with acid showed the disappearance of metabolite 1, 2, 5 and 7 indicating these to be the conjugated metabolites. Further, digestion of urine of 3-BBA treated guinea pigs with glucuronidase showed disappearance of metabolite 5 and 7 suggesting these as glucuronide conjugates. Digestion of urine with sulfatase enzyme resulted in disappearance of metabolite 1 which could be a sulfate conjugate. Urinary metabolite 2 which was found to be present even after digestion with glucuronidase or sulfatase but disappeared following acid treatment appears to be glutathione conjugate(s) which resulted in formation of metabolite 3 and 6. These results suggest that conjugated fluorescent metabolites of 3-BBA are excreted in urine of rats and guinea pigs.

Comparative effect of benzanthrone and 3-bromobenzanthrone on hepatic xenobiotic metabolism and anti-oxidative defense system in guinea pigs.

Benzanthrone (BA) and 3-bromobenzanthrone (3-BBA) are important dye intermediates used in the manufacture of various vat and disperse dyes. BA has been implicated as a cause of hepatic malfunctions and dermal lesions in workers. However, not much information on halogenated BAs, especially 3-BBA, is available. Experiments were designed to undertake a comparative safety assessment of both BA and 3-BBA, given orally at a dose of 50 mg/kg body weight for 10 days to guinea pigs. There was a significant decrease (25%) in body weight with 3-BBA, whereas BA treatment did not cause any change. Serum glutamate oxaloacetate transaminase and glutamate pyruvate transminase were found to be significantly (P<0.05) increased in 3-BBA- as well as in BA-treated animals. 3-BBA and BA led to substantial depletion of ascorbic acid in both liver and adrenal glands. However, depletion of ascorbic acid was more pronounced with 3-BBA (19.2-28.3%) than with BA (13.5-16.6%). 3-BBA and BA treatments caused 80% and 24% depletion of hepatic free sulfydryl content, while lipid peroxidation showed a significant enhancement of 73% and 47%, respectively. BA and 3-BBA caused decreases in cytochrome P-450 content and phase I enzymes particularly ethoxyresorufin- O-deethylase and aryl hydrocarbon hydroxylase, whereas phase II enzymes (quinone reductase and glutathione- S-transferase) were substantially increased. Activities of bio-antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase, were significantly increased by 153, 104, 20 and 67% in the 3-BBA-treated group, whereas the degree of increase in these parameters was relatively less in BA-treated group. The data indicate that both BA and 3-BBA can disturb membrane integrity by decreasing endogenous glutathione and ascorbic acid levels with a concomitant increase in lipid peroxidative damage. This may in turn lead to impairment of hepatic P-450-dependent monooxygenase, while the changes in antioxidant enzymes reveal oxidative stress. 3-BBA treatment caused dilation of portal triad with thickening of arterial wall, hyperplasia of Kupffer cells and influx of inflammatory cells between hepatic cords, which could be due to formation of Br(*) radical or due to formation of semiquinone type of intermediate following oxidation. The results may be interpreted to mean that industrial workers exposed to 3-BBA are at higher risk than those exposed to BA, and necessary precautions should be taken to safeguard their exposure risks.