Treatment of endosulfan contaminated water with in vitro plant cell cultures.

Endosulfan is a Persistent Organic Pollutant insecticide still used in many countries. It is commercially available as mixtures of two diastereomers, α- and ß-endosulfan, known as technical grade endosulfan (TGE). A laboratory model based on the use of axenic plant cell cultures to study the removal and metabolization of both isomers from contaminated water matrixes was established. No differences were recorded in the removal of the two individual isomers with the two tested endemic plants, Grindelia pulchella and Tessaria absinthioides. Undifferentiated cultures of both plant species were very efficient to lower endosulfan concentration in spiked solutions. Metabolic fate of TGE was evaluated by analyzing the time course of endosulfan metabolites accumulation in both plant biomass and bioremediation media. While in G. pulchella we only detected endosulfan sulfate, in T. absinthioides the non-toxic endosulfan alcohol was the main metabolite at 48h, giving the possibility of designing phytoremediation approaches.

Deracemization of secondary alcohols by chemo-enzymatic sequence with plant cells.

A screening based on undifferentiated plant cells allowed identifying Gardenia jasminoides as the best biocatalyst to perform the kinetic resolution of 1-phenylethanol. This species was further tested for its ability to oxidize stereoselectively the (S)-isomers from racemic mixtures of secondary alcohols leaving their antipodes unaffected in Tris-HCl buffer. Those substrates which afforded the best results in the kinetic resolution were subjected to a chemo-enzymatic sequence of deracemization. G. jasminoides immobilized cells in calcium alginate were used for the oxidation of the (S)-enantiomers and, in a second step, NaBH(4) was added to the same vessel for the reduction of the corresponding ketone. The sequential repetition of these two steps allowed obtaining the R-alcohols in 82-90% yield in high optical purity (71-96% ee). Despite the viability of the cells is affected by the chemical reagent, their enzymes remain active due to the protective environment of the calcium alginate beads.

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.