Poplar demand.

Trichloroethylene (TCE) is a common industrial solvent that poses a particular pollution problem in groundwater; while TCE disappears from surface water within a few weeks, groundwater contamination can take months or years to degrade. Humans have not conclusively been shown to develop cancer in response to TCE exposure, but rats and mice exposed to TCE have an increased incidence of liver and lung cancers.

The development of resistance to chemotherapeutic drugs by neoplastic cells is a major obstacle to the cure of many malignancies. Recent reports indicate that it is possible to reverse resistance to vincristine and to daunorubicin in murine tumours in vivo by use of verapamil hydrochloride, the calcium channel blocking agent (Tsuruo et al., 1981;Slater et al., 1982;Xkovsgaard et al., 1984). The use of verapamil in patients with malignancy has, however, been limited by high concentration requirements (Kessel & Wilberding, 1985a). We now describe complete reversal of daunorubicin resistance in daunorubicin resistant Ehrlich ascites carcinoma by cyclosporin A used in doses previously employed in humans.

Materials and methods
Tumour lines and treatment regimens Ehrlich ascites carcinoma (EA) was maintained as an ascitic tumour in BALB/c mice. A daunorubicinresistant subline was developed by sequential transfer of EA cells to subsequent generations of host mice with continuous daunorubicin treatment as previously described (Slater et al., 1982). For current studies, the daunorubicin treatment regimen consists of 0.3mgkg-' daunorubicin i.p. daily for five doses, starting 24h after the inoculation of 0.2ml, i.p. of undiluted malignant ascites harvested from preterminal animals. CsA (Sandimmune I.V., Sandoz LTD) is given i.p. either alone or simultaneously with daunorubicin at the doses indicated.
Samples were collected on glass fibre filters with a Titertek multiple automated sample harvester unit employing a deionized water wash. The filters were dried and counted in a PPO/POPOP/toluene    (Slater et al., 1982). In vivo, the  EA/DS and EA/DR signify daunorubicin sensitive and resistant tumour lines, respectively. The treatment regimen consists of daunorubicin 0.3 mg kgi.p. daily for five doses, starting 24 h after the inoculation of 0.2 ml i.p. of undiluted malignant ascites harvested from preterminal BALB/c host mice. Total CsA treatment regimens given either alone or simultaneously with daunorubicin in five divided doses are indicated; aIndicates the death of one animal in this group at day 5 with diarrhoea. Chi square values compare the frequency of 60 day survival between the indicated groups; bIndicates mean survival calculated using 60 day survival for each long survivor.
addition of CsA to daunorubicin in the treatment of EA/DR bearing host mice also restores responses to daunorubicin. When CsA is added to the daunorubicin treatment of the resistant subline, the reduced survival of host mice bearing daunorubicin resistant EA treated with daunorubicin alone returns to the survival characteristic of host mice bearing daunorubicin sensitive EA.
Although the doses of CsA used in these in vivo murine studies are similar to those previously employed in humans (Biggs et al., 1983;Kennedy et al., 1985) the use of intraperitoneal CsA in the treatment of an ascitic tumour would favour high local drug concentrations. It is of interest, however, that tissue levels of CsA determined at postmortem examination in patients maintained on CsA for over one week until death range from several hundred to over 7000ngg-1 of tissue wet weight (Ried et al., 1983). These values compare favourably to the 3300ngml-' CsA concentration demonstrated to reverse daunorubicin resistance in EA in vitro. Although the efficacy of CsA in reversing drug resistance of human tumours in vivo has not yet been demonstrated, we do note important in vitro activity of CsA in daunorubicin resistant human acute lymphatic leukaemia. CsA completely reverses 50-fold primary resistance to vincristine and 5-fold cross resistance to dauno-rubicin in a pleiotropic drug resistant subline of GM3639 human T cell acute lymphatic leukaemia -Human Genetic Mutant Cell Repository, Camden, New Jersey (Slater et al., 1986).
The mechanism by which CsA restores responses to daunorubicin and to vincristine is unclear, but may relate to its ability to inhibit calmodulin (Colombani et al., 1985). Verapamil and calmodulin inhibitors have been shown to correct the enhanced active efflux of vinca alkaloids and anthracycline antibiotics characteristic of drug resistant tumour cells resulting in increased cellular drug retention (Tsuruo et al., 1981;. However, Kessel & Corbett (1985) were unable to demonstrate a correlation between adriamycin uptake and adriamycin resistance in murine solid tumours rendered resistant to adriamycin by in vivo drug exposure. We were similarly unable to detect significant differences in daunorubicin uptake between our daunorubicin-sensitive and daunorubicin-resistant Ehrlich ascites carcinoma subline and drug sensitive versus pleiotropic drug resistant human acute lymphatic leukaemia subline (Slater et al., 1986), suggesting that the mechanism of CsA effect lies beyond the modification of drug transport. Since the acquisition of equimolar concentrations of anthracyclines by anthracycline resistant compared to anthracycline sensitive tumour cells fails to restore equivalent cytotoxic drug effect to these cells (Kessel & Wilberding, 1985b;Sikic et al., 1985), the mechanism by which calcium channel blocking agents and calmodulin inhibitors restore drug sensitivity must extend beyond drug retention. It has been shown that adriamycin is cytotoxic when the drug is bound to polygluteraldehyde microspheres or to agarose, which prevents its entry into tumour cells and suggests that cytotoxicity occurs as a cell surface phenomenon (Tritton & Yee, 1982;Rogers et al., 1983). The mechanism of anthracycline antibiotic resistance relates to the cell membrane as well, since recent reports show that pleiotropic drug resistant cells possess characteristic cell membrane glycoprotein alterations in addition to the enhanced active drug efflux described above (Kartner et al., 1983a,b).
The studies of LeGrue et al. (1983) raise the possibility that CsA might function in the plasma membrane in a manner similar to that of lipid soluble anaesthetics by increasing lipid fluidity and uncoupling electrochemical action potentials. Since drug retention alone cannot account for the effects of calcium channel blocking drugs and calmodulin inhibitors on vinca alkaloid and anthracycline antibiotics in pleiotropic drug resistant cells, it has been suggested that these agents, and it now appears that CsA, may enhance intracellular drug binding or promote favourable chemotherapeutic drug interactions at the membrane level (Beck, 1983;Kessel & Wilberding, 1985b).