Subhash K. Khanna

Detection of Aflatoxin M1 contamination in milk and infant milk products from Indian markets by ELISA

Abstract The occurrence of Aflatoxin M1 (AFM1) contamination in Indian infant milk products and liquid milk samples was investigated by competitive ELISA technique. A total of 87 samples in categories of infant milk food (18), infant formula (17), milk based cereal weaning food (40) and liquid milk samples (12) showed that the incidence of contamination of AFM1 was of the magnitude of 87.3%. The range of contamination of AFM1 was comparatively higher in infant milk products (65–1012 ng/l) than liquid milk (28–164 ng/l). Almost 99% of the contaminated samples exceeded the European Communities/Codex Alimentarius recommended limits (50 ng/l), while 9% samples exceeded the prescribed limit of US regulations (500 ng/l). The extrapolation of AFM1 data to estimate the Aflatoxin B1 (AFB1) contamination in dairy cattle feedstuffs indicate that the contamination may range from 1.4 to 63.3 μg/kg with a mean of 18 μg/kg which is substantially higher than the directive of European Communities regulation (5 μg/kg). The results suggest a need to introduce safety limits for AFM1 levels (480 ng/kg) in infant milk products and liquid milk under Prevention of Food Adulteration Act of India as well as to prescribe the levels of AFB1 in dairy cattle feedstuffs so as to minimize the health hazard risk in infant population at large.

Clinicoepidemiological, Toxicological, and Safety Evaluation Studies on Argemone Oil

Consumption of oil extracted from accidental or deliberate contamination of argemone seed to mustard seed is known to pose a clinical condition popularly referred to as Epidemic Dropsy. Several outbreaks of Epidemic Dropsy have occurred in the past in India as well as in Mauritius, Fiji Island, and South Africa. Clinico-epidemiological manifestations of argemone oil poisoning include vomiting, diarrhea, nausea, swelling of limbs, erythema, pitting edema, breathlessness, etc. In extreme cases, glaucoma and even death due to cardiac arrest have been encountered. The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Histopathological studies suggest that liver, lungs, kidney, and heart are the target sites for argemone oil intoxication. Studies have shown to elucidate the cocarcinogenic potential of argemone oil that can be correlated with the binding of sanguinarine with a DNA template. Pharmacological response in intestine revealed immediate stimulation of tone and peristaltic movements of the gut in the sanguinarine-treated animals. Argemone oil/Sanguinarine caused a decrease in hepatic glycogen levels which may be due to the activation of glycogenolysis leading to an accumulation of pyruvate in the blood of Epidemic Dropsy cases. The increase in pyruvate levels causes uncoupling of oxidative phosphorylation leading to breathlessness, as observed in patients. Sanguinarine has been shown to inhibit Na+, K(+)-ATPase activity of different organs such as brain, heart, liver, intestine, and skeletal muscle, which may be due to the interaction with the glycoside receptor site on ATPase enzyme, thereby causing a decrease in the active transport of glucose. Argemone oil/alkaloid showed a Type II binding spectra with hepatic cytochrome P-450 (P-450) protein, thereby causing loss of P-450 content and an impairment of phase I and phase II enzymes. A green fluorescent metabolite of sanguinarine, benzacridine was detected in the milk of grazing animals. The delayed appearance of this metabolite in urine and feces of experimental animals suggests the slow elimination of the alkaloid. Argemone oil enhances hepatic microsomal and mitochondrial lipid peroxidation, indicating that these two organelles are the sites of membrane damage. Furthermore, studies suggest that singlet oxygen and hydroxyl radical are involved in argemone oil toxicity. Several bioantioxidants show protective effect in argemone oil-induced toxicity in experimental animals. The line of treatment in argemone-intoxicated epidemics has so far been only symptomatic, and specific therapeutic measures are still lacking, although it has been suggested that diuretics, bioantioxidants, steroids, vitamins, calcium- and protein-rich diet had some beneficial effects on Epidemic Dropsy cases.

Role of antioxidants and scavengers on argemone oil-induced toxicity in rats.

The role of antioxidants and scavengers on argemone oil-induced enzymatic and non-enzymatic hepatic lipid peroxidation was investigated in rats. Multiple treatment of argemone oil caused a significant stimulation of NADPH-dependent enzymatic or FeSO4 or FeSO4/ADP- or ascorbic acid-dependent non-enzymatic hepatic microsomal lipid peroxidation.In vitro addition of antioxidants such as tannic acid, quercetin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, riboflavin or glutathione (GSH) in the assay system resulted in significant protection against argemone oil-induced microsomal NADPH, or FeSO4/ADP-dependent lipid peroxidation.In vitro addition of scavengers of the superoxide anion (O2−) radical and hydrogen peroxide (H2O2) such as superoxide dismutase (SOD) and catalase, respectively, prevented argemone oil augmented microsomal lipid peroxidation to a lesser extent as compared to the scavengers of singlet oxygen (1O2) such as 2,5-dimethylfuran (DMF), β-carotene, and histidine or hydroxyl (OH•) radical scavengers such as ethanol, mannitol and sodium benzoate. These results suggest that primarily1O2 and OH• radicals are involved in argemone oil-induced hepatic microsomal lipid peroxidation, and that bio-antioxidant vitamins including riboflavin, β-carotene and α-tocopherol may prove useful in reducing argemone oil-induced hepatotoxicity.

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 μmol) 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 γ‐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.

Role of Cytochrome P-450 in Quinalphos Toxicity: Effect on Hepatic and Brain Antioxidant Enzymes in RatsITRC Communication No. 1965.

Quinalphos (QP), an organophosphate pesticide, is used in controlling the pests of a variety of crops. To understand the mechanism of the metabolic basis of the toxicity of QP it was thought pertinent to study the role of cytochrome P-450 (P450) and antioxidant enzyme systems. Albino rats treated orally with QP (0.52 and 1.04 mg/kg body weight) for 60 days showed a significant decrease in body, brain and liver weights. Hepatic P450 content and its dependent monooxygenases, namely aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (ERD), were induced to 1.8-2.5-fold, while neuronal AHH was induced to 1.8-fold following QP treatment (1.04 mg/kg) to animals. The hepatic antioxidant defence system, comprising catalase, glutathione (GSH) reductase, superoxide dismutase (SOD) and GSH peroxidase, was also significantly increased in QP-treated animals, while in the brain only catalase was increased and GSH reductase decreased. There was no significant change in hepatic GSH content and lipid peroxide levels in QP treated animals at any dose group in comparison with the control group. Pretreatment of rats with phenobarbitone (PB) or 3-methylcholanthrene (MC) (P450 inducers) prevented mortality caused by the LD50 dose of QP, whereas pretreatment with cobalt chloride (a P450 inhibitor) enhanced the mortality rate to 100% within 3 days. From the above study it can be inferred that the toxicity of QP may be due to the parent compound or its metabolite(s) produced prior to P450 oxidation and that the induction of P450 system by QP may be a defence mechanism.

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.

Biochemical toxicology of argemone oil. IV short-term oral feeding response in rats

Consumption of edible oils contaminated with Argemone mexicana seed oil is known to cause various clinical manifestations. In the present study, the effect of dietary intake of argemone oil on histopathological changes, haematological indices and selected marker parameters of toxicity was investigated to observe the exact sites and mode of action of argemone oil in rats. Histopathological changes in the liver showed increased fibrosis, hyperplasia of bile ducts and congestion in a few portal tracts. Lungs of argemone oil-fed animals indicated congestion and thickening of interalveolar septa. Alveolar spaces were disorganised and irregular. Kidneys showed vascular and glomerular congestion and patchy tubular lesions. At 30 days only mild congestion was noted in the myocardium. Cardiac muscle fibres showed degenerative changes at 60 days which were more marked in the auricular wall. Haematological examination showed appearance of anaemia in experimental animals. Hepatic alkaline phosphatase, alanine transaminase and aspartate transaminase activities were inhibited by 30, 29 and 29% after 30 days of argemone intake along with concomitant enhancement in serum by 27, 29 and 66%, respectively. Liver showed decrease in glutathione (32-63%) content along with significant stimulation of lipid peroxidation (49-105%) in argemone-intoxicated animals. These results suggest that liver, lungs, heart and kidneys are the target tissues of argemone oil toxicity and that membrane destruction may be a possible mode of action.

Biochemical toxicology of argemone oil. Role of reactive oxygen species in iron catalyzed lipid peroxidation

Contamination of mustard oil with the resembling seed oil of the weed Argemone mexicana L., is quite common in India. Consumption of such contaminated oil even for short duration causes a clinical condition popularly termed as Epidemic Dropsy

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.

Surveillance of the quality of turmeric powders from city markets of India on the basis of curcumin content and the presence of extraneous colours

Curcumin, the principal curcuminoid of turmeric, is responsible for its yellow colour and serves as a measure of turmeric quality. The Prevention of Food Adulteration (PFA) Act of India allows only Curcuma longa L. for the production of turmeric powder and prohibits addition of any foreign matter/artificial colour, but it does not specify a minimum curcumin content. The present surveillance was undertaken to study the quality of loose versus branded turmeric powders vis-à-vis curcumin content and the presence of unwarranted extraneous colours from city markets in India using a newly developed two-dimensional high-performance thin-layer chromatography (2D-HPTLC) method. The results show that curcumin content in branded samples ranged from 2.2% to 3.7%, while non-branded samples had from 0.3% to 2.6%. Though none of the branded turmeric powders contained artificial colours, 17% of loose powders showed the presence of extraneous colour metanil yellow, in the range 1.0–8.5 mg g−1, which may pose health threats. Low curcumin content in the analysed samples may be due to mixing of other curcuma species or their curcumin-depleted matrices and foreign starches as cheaper alternatives. This is supported by the fact that major Indian turmeric trade types are known to possess curcumin contents ranging from 2.1% to 8.6%, with an average of 4.8%. There is thus an urgent need to prescribe realistic curcumin limits for turmeric powder, otherwise there is no obligation on the part of traders to stick to any minimum levels and consumers will keep on getting this nutrient-depleted household spice.