Heterophyllin: A New Adenia Toxic Lectin with Peculiar Biological Properties.

Ribosome-inactivating proteins (RIPs) are plant toxins that were identified for their ability to irreversibly damage ribosomes, thereby causing arrest of protein synthesis and induction of cell death. The RIPs purified from Adenia plants are the most potent ones. Here, we describe a novel toxic lectin from Adenia heterophylla caudex, which has been named heterophyllin. Heterophyllin shows the enzymatic and lectin properties of type 2 RIPs. Interestingly, in immunoreactivity experiments, heterophyllin poorly cross-reacts with sera against all other tested RIPs. The cytotoxic effects and death pathways triggered by heterophyllin were investigated in three human-derived cell lines: NB100, T24, and MCF7, and compared to ricin, the most known and studied type 2 RIP. Heterophyllin was able to completely abolish cell viability at nM concentration. A strong induction of apoptosis, but not necrosis, and the involvement of oxidative stress and necroptosis were observed in all the tested cell lines. Therefore, the enzymatic, immunological, and biological activities of heterophyllin make it an interesting molecule, worthy of further in-depth analysis to verify its possible pharmacological application.

Fruticulosin: A novel type 2 ribosome-inactivating protein from Abrus fruticulosus seeds that exhibits toxic and antileishmanial activity.

Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60 kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), ß-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12 µg/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major.

Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions).

Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications.

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea's defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

Cytotoxicity mechanism of α-MMC in normal liver cells through LRP1 mediated endocytosis and JNK activation.

Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein isolated from Momordica charantia, is a potential drug candidate with strong anti-tumor activity. However, α-MMC has a severe hepatotoxicity when applied in vivo, which may greatly hinders its use in clinic in the future. The biological mechanism of hepatotoxicity induced by α-MMC is largely unknown, especially the mechanism by which α-MMC enters the hepatocytes. In this study, we investigated α-MMC-induced cytotoxicity in normal liver L02 cell line as well as the mechanism underlying it. As expected, α-MMC is more toxic in L02 cells than in various normal cells from other organs. The cytotoxic effect of α-MMC on L02 cells is found to be mediated through cell apoptosis as detected by flow cytometry and fluorescence microscopy. Importantly, α-MMC was shown to bind to a specific receptor on cell membrane, as the density of the cell membrane receptor is closely related to both the amount of α-MMC endocytosed and the cytotoxicity in different cell lines. By using LRP1 competitive inhibitor α2-M or siRNA targeting LRP1, we further identified that LRP1 protein served as the membrane receptor for α-MMC. Both α2-M and siRNA targeting LRP1 can significantly inhibit α-MMC's endocytosis as well as its cytotoxicity in L02 cells. In addition, it was found that α-MMC can activate the JNK signalling pathways via LRP1 in L02 cells. As JNK activation often leads to cell apoptosis, the activation of JNK may play an important role in α-MMC-induced cytotoxicity. To our knowledge, this is the first report showing that LRP1 mediates the cytotoxicity of α-MMC through (1) endocytosis and induced apoptosis and (2) the activation of the JNK pathway. Our findings shed light on the fundamental mechanism of hepatotoxicity of α-MMC and offer reference to understand its mechanism of lymphocytotoxicity and neurotoxicity.

PEGylation alleviates the non-specific toxicities of Alpha-Momorcharin and preserves its antitumor efficacy in vivo.

Alpha-Momorcharin (α-MMC) is a ribosome inactivating protein from Momordica charantia with anti-tumor activity. Previously, we had observed that modification of α-MMC with polyethylene glycol (PEG) could reduce toxicity, but it also reduces its anti-tumor activity in vitro. This study aims to investigate whether the metabolism-extended properties of α-MMC resulting from PEGylation could preserve its anti-tumor efficacy in vivo through pharmacokinetics and antitumor experiments. The pharmacokinetics experiments were conducted in rats using the TCA (Trichloroacetic Acid) method. Antitumor activity in vivo was investigated in murine mammary carcinoma (EMT-6) and human mammary carcinoma (MDA-MB-231) transplanted tumor mouse models. The results showed that PEGylation increased the plasma half-life of α-MMC in rats from 6.2-7.5 h to 52-87 h. When administered at 1 mg/kg, α-MMC-PEG and α-MMC showed similar anti-tumor activities in vivo, with a T/C% of 38.56% for α-MMC versus 35.43% for α-MMC-PEG in the EMT-6 tumor model and 36.30% for α-MMC versus 39.88% for α-MMC-PEG in the MDA-MB-231 tumor model (p > 0.05). Importantly, at the dose of 3 mg/kg, all the animals treated with α-MMC died while the animals treated with α-MMC-PEG exhibited only moderate toxic reactions, and α-MMC-PEG exhibited improved anti-tumor efficacy with a T/C% (relative tumor growth rate) of 25.18% and 21.07% in the EMT-6 and MDA-MB-231 tumor models, respectively. The present study demonstrates that PEGylation extends the half-life of α-MMC and alleviates non-specific toxicity, thereby preserving its antitumor efficacy in vivo, and a higher lever of dosage can be used to achieve better therapeutic efficacy.

Alpha-momorcharin (α-MMC) exerts effective anti-human breast tumor activities but has a narrow therapeutic window in vivo.

Alpha-momorcharin (α-MMC), a ribosome inactivating protein (RIP) extracted from the seeds of Momordica charantia, exerts anti-tumor, antiviral, and anti-fungal activities. However, α-MMC has an obvious toxicity that limits its clinical application. We examined the effect of α-MMC on the inhibition of human breast cancer and assessed its general toxicity to find the therapeutic window in vivo for its potential clinical use. It was purified using column chromatography, and then injected into the xenograft nude mouse model induced by MDA-MB-231 and MCF-7. The anti-tumor efficacy was evaluated with T/C%. Next, the α-MMC was injected at a series of doses to Balb/C mice to assess its general toxicity. The MTT assay, the apoptosis test, and the cell cycle inhibition of α-MMC in human breast cancer cells were performed. In the xenografted tumors induced by MDA-MB-231 and MCF-7, α-MMC exerted an obvious inhibition effects on tumor growth at the dosage of 1.2mg/kg and 0.8 mg/kg. For in vivo toxicity experiments of α-MMC in Balb/C mice, the minimal toxic dose of α-MMC was 1.2mg/kg. Alpha-MMC induced apoptosis by increasing caspase3 activities, and the cell cycle was arrested at the G0/G1 or G2/M phases. The measurements of IC50 were 15.07 µg/mL, 33.66 µg/mL, 42.94 µg/mL for MDA-MB-231, MCF-7 and MDA-MB-453 respectively. Alpha-MMC exhibits anti-tumor effects in human breast cancer in vivo and in vitro. It inhibits breast cancer cells through the inhibition of tumor growth and induction of cell apoptosis. However, due to its obvious toxicity, α-MMC has a relatively narrow therapeutic window in vivo.

Paneth cells are also target of the ribotoxic lectin nigrin b.

Ribosome-inactivating lectins (RILs) are A-B type toxins like ricin whose molecular target is the large rRNA of eukaryotic ribosome. Administration of lethal doses of the RIL nigrin b isolated from elderberry (Sambucus nigra L.) bark triggers specific intestinal derangement. The aim of the present research was to explore the early effects of a lethal dose of nigrin b (16 mg/kg body weight) on the small intestine using light and electron microscopy to ascertain intestinal epithelium changes. 6 h after nigrin administration, the small intestine crypts began to show signs of damage with cells appearing at different stages of apoptosis. 16 h after injection crypts appeared more impaired, including the derangement of Paneth cells. The novelty of our results is that the Paneth cells in the small intestine in addition to stem cells are the early cellular targets for nigrin b.

[Effect of PEGylation of alpha-Momorcharin against its hepatotoxicity in rats].

OBJECTIVE: To explore the effect of PEGylation of alpha-Momorcharin (alpha-MMC), one of ribosome-inactivating proteins from bitter melon seed, against its hepatotoxicity in rats. METHODS: SD rats were randomized into NS group, alpha-MMC treated groups, and alpha-MMC-PEG treated groups. The doses of alpha-MMC and alpha-MMC-PEG were high, middle, and low dose (6.25, 2.08, 0.70 mg/kg). The rats were given different dose of alpha-MMC, or alpha-MMC-PEG via caudal vein every other day for consecutive 28 days and then left for 14 days recovery. The general condition of animals was observed, blood and liver samples were collected for liver function study and pathological examination on day 28 after initiation of administration and on day 14 after withdrawal. RESULTS: On day 28 after initiation of administration, the liver function damages were found in high-dose and middle-dose of alpha-MMC treated groups, such as the decreasing of ALB, increasing of GLB, A/G ratio decreasing and the dose-dependant increasing of AST, BIL and CHO. The pathological changes of hepatotoxicity were also observed in these two groups, including the massive hepatocyte, swelling degeneration, inflammatory cell infiltration, congestion and diffusive necrosis. However, the liver function and pathological changes in alpha-MMC-PEG treated groups were better than those in alpha-MMC treated groups. CONCLUSION: PEGylation could reduce the hepatotoxicity of alpha-MMC to rats.

Ribosome-inactivating proteins: potent poisons and molecular tools.

Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

Ribosome-inactivating proteins: from toxins to useful proteins.

Ribosome-inactivating proteins (RIPs) either single-chain (type 1) or two-chain (type 2) are frequent in plants, often in multiple forms. They are RNA N-glycosidases, have antiviral, antifungal and insecticidal activity. Their expression in plants is increased under stressful conditions. They are investigated for practical applications in medicine and in agriculture. In medicine, RIPs have been linked to, or fused with, appropriate antibodies or other carriers to form "immunotoxins" or other conjugates specifically toxic to the cells target of the carrier, with the aim of eliminating malignant or other undesired cells. In agriculture, it has been observed that an enhanced expression of RIPs confers to plants an increased resistance to viruses, fungi, insects, and also to drought and salinity.

Ribosome-inactivating proteins with an emphasis on bacterial RIPs and their potential medical applications.

Ribosome-inactivating proteins (RIPs) are toxic due to their N-glycosidase activity catalyzing depurination at the universally conserved α-sarcin loop of the 60S ribosomal subunit. In addition, RIPs have been shown to also have other enzymatic activities, including polynucleotide:adenosine glycosidase activity. RIPs are mainly produced by different plant species, but are additionally found in a number of bacteria, fungi, algae and some mammalian tissues. This review describes the occurrence of RIPs, with special emphasis on bacterial RIPs, including the Shiga toxin and RIP in Streptomyces coelicolor recently identified in S. coelicolor. The properties of RIPs, such as enzymatic activity and targeting specificity, and how their unique biological activity could be potentially turned into medical or agricultural tools to combat tumors, viruses and fungi, are highlighted.

Alpha-momorcharin possessing high immunogenicity, immunotoxicity and hepatotoxicity in SD rats.

UNLABELLED: Momordica charantia L., a genus of Momordica Linn. of the family Cucurbitaceae, commonly known as bitter melon, has been widely planted in China, Southeast Asia, Turkey and other areas, and has been used as a medicine for a long time. Alpha-momorcharin (α-MMC) extracted and purified from bitter melon seeds has significant anti-tumor and anti-virus effects, and has potential toxicity as well, especially when taken overdose. However, up to date studies on its safety evaluation are still insufficient. AIMS OF THE STUDY: The immunogenicity, immunotoxicity and general toxicity of α-MMC were investigated in rats and guinea-pigs, and the potential toxic effects of the agent on the body were also examined. MATERIALS AND METHODS: The major ribosome-inactivating protein was isolated by column chromatographies from the protein extracted from bitter melon seeds, and was verified as α-MMC. After rats were immunized by α-MMC, titers of specific antibody to α-MMC in immunized rats serum were detected by indirect ELISA. Guinea-pigs and rats immunized with α-MMC were used to evaluate the active systemic anaphylaxis and passive cutaneous anaphylaxis induced by α-MMC relatively. α-MMC of 6.25 mg/kg, 2.08 mg/kg and 0.70 mg/kg was administered to rats every 2 days. Five weeks later, animals were sacrificed, and then, biochemical examination, analysis of bone marrow and peripheral blood cells, and histopathologic examination were performed. RESULTS: The ribosome-inactivating protein isolated and purified from bitter melon seeds was identified as α-MMC. It induced high titer (1:46.4) of specific IgG and high positive results of the active systemic anaphylaxis and passive cutaneous anaphylaxis tests in animals. With the time of the α-MMC administration increasing, the body weights of the animals administered with α-MMC of 6.25 mg/kg decreased significantly, and point necrosis was also observed in liver cells, along with abnormal findings in serum chemistry, hematology and bone marrow histopathology test. The toxic effect lessened with the decrease of the dose of α-MMC and further reduced after the convalescence stage. CONCLUSIONS: The results of the study show that α-MMC has high immunogenicity and immunotoxicity, and can cause obvious organic liver lesion.

RNA toxins: mediators of stress adaptation and pathogen defense.

RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

Entomotoxic action of Sambucus nigra agglutinin I in Acyrthosiphon pisum aphids and Spodoptera exigua caterpillars through caspase-3-like-dependent apoptosis.

In this project, the toxicity and mechanism of action of the ricin-B-related lectin SNA-I from elderberry (Sambucus nigra) in the pea aphid (Acyrthosiphon pisum) and the beet armyworm (Spodoptera exigua), two important pest insects in agriculture, were studied. SNA-I is a chimeric lectin belonging to the class of ribosome-inactivating proteins and consists of an A-chain with N-glycosidase activity and a carbohydrate-binding B-chain. Incorporation of 2 mg/ml of SNA-I in the diet of neonates and adults of A. pisum caused 40-46% mortality within 2 days, while in third instars of S. exigua, the larval biomass was significantly reduced by 12% after feeding for 3 days on a diet containing 5 mg/g of SNA-I. Interestingly, extracts of the (mid)gut of treated A. pisum and S. exigua demonstrated DNA fragmentation and this was accompanied with an increase in caspase-3-like activity. The involvement of cell death or apoptosis in the entomotoxicity of SNA-I through induction of caspase-3-like activity was also confirmed by addition of the permeable caspase-3 inhibitor III in the diet, leading to a rescue of the treated aphid neonates. Finally, similar to the chimeric lectin SNA-I, the hololectin SNA-II, consisting of two carbohydrate-binding B-chains caused high mortality to neonate A. pisum aphids with an LC50 of 1.59 mg/ml, suggesting that the entomotoxic action of the lectins under study mainly relies on their carbohydrate-binding activity.

PEGylation of alpha-momorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential.

Alpha-momorcharin (alpha-MMC) is a ribosome-inactivating protein (RIP) with excellent cytotoxicity to tumor cells. However, its strong immunogenicity and short plasma half-life limit its clinical applications. To overcome this, we have to PEGylated alpha-MMC using a branched 20 kDa (mPEG) (2)-Lys-NHS. Homogeneous mono-, di- and tri-PEGylated alpha-MMCs were synthesized, purified and characterized. In vitro and in vivo analysis indicated that the serial PEG-conjugates preserved moderate anti-tumor activity with 36% acute toxicity and at most 66% immunogenicity decrease. These results suggested the potential application of alpha-MMC-PEG conjugates as an anti-tumor agent.

Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos.

The teratogenicity of two fungal ribosome-inactivating proteins, hypsin from Hypsizigus mamoreus and velutin from Flammulina velutipes, was examined in this investigation using microinjection and postimplantation whole-embryo culture. The results demonstrated that hypsin induced abnormal embryonic development at 2.5 microM during the organogenesis period from E8.5 to E9.5. As its dosage increased, there was an increase in the total number of abnormal embryos, a drop in the final somite number, and a rise of abnormal structures. Structural abnormalities were detected: open cranial neural tube, abnormal branchial arches, absence of forelimb buds and twisted body axis. The otic and optic placodes were, however, less affected. Histological study of the abnormal embryos revealed a correlation of increased cell death with abnormal structures, suggesting that induction of cell death by hypsin may account for its teratogenicity. In contrast, velutin did not exert any adverse influence on mouse development.

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro.

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 microg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 microg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.

[Study on the immunotoxicity of ribosome inhibiting protein of Momordica charantia].

OBJECTIVE: To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. METHODS: Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD50). Active systemic anaphylaxis(ASA)test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. RESULTS: The LD50 (iv) in mice of RIP was 25.2 mg/kg in ASA, guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA, obvious blue dye in skin were observed in SD rats of the RIP group. CONCLUSION: RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine.

Abrin is a plant glycoprotein toxin, classified as ribosome inactivating protein (RIP) due to its property of damaging ribosomes in an irreversible manner. Many RIPs have direct DNA damaging activity. The objective of the present study was to evaluate the oxidative stress and DNA damaging potential of abrin in U937 human myeloleukemic cells. Cells were treated with abrin at IC50 of 8 ng/ml for 24h. Abrin induced a time dependent increase in reactive oxygen species and levels of antioxidant enzymes. There was significant depletion of reduced glutathione levels. DNA damage was assessed by comet assay in terms of percent head DNA, tail DNA, tail length and Olive tail moment. DNA damage was more pronounced at 4 and 8h at IC50 concentration. Abrin at 4, 8, 16 and 32 ng/ml concentration induced significant DNA damage at 4h. There was time dependent increase in levels of 8-OHdG in abrin treated cells indicating the oxidative stress mediated DNA damage. N-Acetylcysteine pretreatment at 10nM for 1h, considerably reversed the abrin induced DNA damage at 16 and 32 ng/ml. Our results clearly show oxidative DNA damage potential of abrin at low concentration.