Raj Khanna

Edible oil adulterants, argemone oil and butter yellow, as aetiological factors for gall bladder cancer

Carcinogenic potential of argemone oil (AO) and butter yellow (BY), the adulterants encountered in edible oil, in gall bladder of Swiss albino mice was undertaken to investigate the potential aetiological factors of gall bladder carcinoma (GBC) in the Indo-Gangetic basin. Twice weekly intraperitoneal (ip) administration of AO (5 ml/kg body wt) and BY (25 mg/kg body wt) to Swiss albino male and female mice for 30 and 60 days indicated that females were more vulnerable to these adulterants in terms of responses to inflammatory markers. Subsequent experiments with dietary exposure of AO (1%) and BY (0.06%) for 6 months in female mice showed symptoms related to cachexia, jaundice and anaemia. High levels of total cholesterol, low density lipoprotein (LDL), TG, bilirubin and low level of high density lipoprotein (HDL) as well as gallstone formation was shown by AO exposure only, leading to the development of adenocarcinoma. BY exposure resulted in adenoma and hyperplasia without stone formation. The cyclooxygenase (COX-2) overexpression was found to be related to prostaglandin E2 (PGE2) production in AO treated mice but not in BY exposed animals, thereby indicating a differential pathway specific carcinogenicity. PGE2 stimulates the secretion of secreted mucins (MUC5AC), which is involved in stone formation following AO exposure. Enhanced secretion of membrane bound mucins (MUC4) in BY and AO exposed mice resulted in the activation of ErbB2 and downstream signalling such as p-AKT, p-ERK and p-JNK, which ultimately affects the target proteins, p53 and p21 leading to adenoma and adenocarcinoma, respectively. The study suggests that AO and BY are responsible for producing GBC in mice along with stone formation in the AO exposed animals.

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.

Evaluation of Dermal Irritancy Potential of Benzanthrone-Derived Dye Analogs: Structure Activity Relationship

The twelve structural analogs of benzanthrone-derived dyes of commercial use were screened for their dermal irritation potential response using the Draize occlusive patch test. The test dyes, dissolved in DMSO as vehicle, were topically applied on the skin of the male Druckery rats (160 ± 10 g) according to the OECD protocol. The potential dermal hazard was assessed in terms of the primary cutaneous irritation (PCI) index and irritancy. Irritancy was evaluated according to the AFNOR scale. In terms of irritancy, the twelve benzanthrone dyes qualified as moderately irritant (3.0–5.0) according to the above scale. In decreasing order, PCI index of the various dyes was: Navy Blue R (4.5); Jade Green XBN (4.25); 16, 17-dihydroxydibenzanthrone (3.84); Black NB (3.75), Jade Green 2G (3.75); 3-bromobenzanthrone (3.58); Brilliant Purple 4R (3.58); Olive D (3.50); Dark Blue 2R (3.41); Olive Green B (3.33); isodibenzanthrone (3.33), and benzanthrone (3.16). These results indicate that benzanthrone-derived dyes/dye intermediates caused dermal toxicity which appears to be influenced by the number of carbonyl and amino-anthraquinone groups as well as by the presence of functional groups like halogen, nitro, hydroxy and methoxy in the parent molecule, benzanthrone.

Argemone Oil and Butter Yellow Induced Toxicity in Hepatic and Extra Hepatic Tissues

Objective: The present study was designed to evaluate the toxicological potential of argemone oil (AO) and butter yellow (BY). Methods: Short term treatment through intraperitoneal administration in hepatic tissue and long term treatment through diet in hepatic and extra hepatic tissues was performed in mice. Results: Short term study showed that female mice were more prone towards risk associated with liver damage than the males. Further investigations in female mice given AO (1%) and BY (0.06%) through diet for 180 days, showed significant weight loss and increase in liver weight. Phase I and Phase II enzymes, antioxidant enzymes and glutathione content were significantly decreased with concomitant increase in lipid peroxidation (LPO) in AO and BY treated mice. Animal fed with AO and BY showed profuse hyperplasia along with fluid filled spaces and patches of hemorrhage in hepatic tissue. AO treated animals showed tumorigenic growth, while BY treatment caused multiple nodule formation in hepatic tissue. Other organs like heart, lungs, kidney and spleen also showed substantial histopathological changes. Conclusion: The results suggest that AO and BY treatment may cause oxidative stress and inhibited phase I and II enzymes leading to accumulation of parent compound(s) or their metabolites, which may result in the tumorigenic/toxic responses in hepatic and extra hepatic tissues.

A SENSITIVE METHOD OF MONITORING EXPOSURE TO 3-BROMOBENZANTHRONE IN INDUSTRIAL DYESTUFF WORKERS

3-Bromobenzanthrone (3-BBA) is an anthraquinone dye intermediate widely used for the synthesis of a variety of dyes. The monitoring of 3-BBA exposure in dyestuff industry workers has not been possible until now as no procedure has been available. In this article, the fluorescence properties of 3-BBA has been utilized to develop a quantitative method for the detection of this dye intermediate. The procedure allows the measurement of trace quantities of 3-BBA in biological specimens, including urine, serum, liver, and feces, in experimental studies. The method involves extraction of biological samples with an equal volume of a chloroform-methanol mixture (1:1, v/v) followed by measurement of the relative fluorescence intensity at excitation maxima of 400 nm and emission maxima of 530 nm. The detection limit was found to be as low as 50 ng of 3-BBA. The procedure can be routinely used to screen for the presence of 3-BBA in biological fluids, especially urine, as a measure of exposure to 3-BBA in dyestuff workers. The authors are thankful to Dr. P. K. Seth, Director, Industrial Toxicology Research Centre, for his keen interest in this study. The secretarial assistance of Mr. K. G. Thomas is duly acknowledged. RPS is thankful to University Grant Commission, New Delhi, for the award of a Senior Research Fellowship.

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.