Apoptosis of Lymphocytes in the Presence of Cr(V) Complexes: Role in Cr(VI)-Induced Toxicity Chellappa Vasant, Kuppusamy Balamurugan, Rama Rajaram, 1 and Thirumalachari Ramasami 1 Central Leather Research Institute, Adyar, Chennai 600 020, India Received June 19, 2001 Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through ep- idemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxida- tion states such as Cr(V) and Cr(IV) in Cr(VI)- induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human pe- ripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll–Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)- oxochromate(V), Na[Cr V O(ehba) 2 ], 1 and sodium bis(2- hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr V O- (hmba) 2 ], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time- and concentration- dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before expo- sure to chromium(V) complexes reverse apoptosis par- tially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible. © 2001 Academic Press Key Words: Cr(V) complexes; apoptosis; DNA frag- mentation; antioxidants; toxicity; Cr(VI); carcino- genesis. Epidemiological studies have shown that several compounds of chromium(VI) used in diverse industries express mutagenic and carcinogenic effects (1, 2). Such genotoxicity studies investigated have included dem- onstration of chromosomal aberrations, apoptosis and cell transformation (3–5). Generally the genotoxic ef- fects of Cr(VI) are attributed to a facile anionic trans- portation of Cr(VI) into the intracellular compartment followed by reaction with intracellular reductants such as ascorbate, glutathione, cysteine, and NADPH, yield- ing Cr(III) through the formation of reactive interme- diate oxidation states such as Cr(V) and Cr(IV) (6 –10). Since these intermediates are highly reactive and la- bile, their role in the carcinogenicity of Cr(VI) is being emphasized in recent times. Formation of other inter- mediates such as organic radicals and reactive oxygen species has also been demonstrated (11, 12). Earlier studies have argued that Cr(III) may well be the ulti- mate carcinogenic form of Cr(VI). This argument was based on the demonstration that DNA–DNA and DNA–protein cross-links could be initiated in the pres- ence of Cr(III) (13–16). In recent times, the emphasis on the role of unusual oxidation states like Cr(V) and Cr(IV) in causing damage to DNA resulting in biotox- icity has attracted attention. When a mice is injected with Cr(VI), in vivo formation of Cr(V) has been dem- onstrated using L-band EPR study (17). When ascor- bate is used as a reductant for Cr(VI), EPR detectable Cr(V) has been observed and it is correlated with the relaxation of plasmid DNA (18). Formation of Cr(V)– glutathione complex involved in the formation of GSH– Cr–DNA adduct, abasic sites and single strand breaks on DNA have also been discussed (19 –21). Earlier studies in our laboratory have indicated that, while some Schiff base complexes of Cr(III) such as trans- diaqua[1,2-bis(salicylideneamino)ethane]chromium(III) perchlorate (Cr(III)salen) and trans-diaqua[1,3-bis- (salicylideneamino)propane]chromium(III) perchlorate (Cr(III)salprn) induce apoptosis of lymphocytes in cell cul- tures, diaquaethylenediaminetetraacetatochromium(III) (Cr(III)EDTA) and potassium trisoxalatochromiate(III) (Cr(III)oxalate) does not elicit any such effects (22). The accessibility of Cr(V) states in the Schiff base ligand environments has been suggested as a possible cause of apoptosis elicited by these Cr(III) complexes 1 To whom correspondence may be addressed. Fax: +91 044 4912150. E-mail: ramarajaram@hushmail.com. Biochemical and Biophysical Research Communications 285, 1354 –1360 (2001) doi:10.1006/bbrc.2001.5335, available online at http://www.idealibrary.com on 1354 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.