Plasma glutamine depletion and patient outcome in acute ICU admissions.

OBJECTIVE: To evaluate whether low plasma glutamine (PG) is related to severity of illness, and actual and predicted hospital mortality. DESIGN: Prospective cohort study. SETTING: 18-bed closed format general intensive care unit (ICU) of a teaching hospital. PATIENTS: Cohort of 80 seriously ill patients non-electively admitted to the ICU. INTERVENTIONS: Blood sampling for the determination of PG at ICU admission. MEASUREMENTS AND RESULTS: Severity of illness and predicted mortality were calculated using the locally validated APACHE II, SAPS II, and MPM II 0 and 24 systems. Illness scores, and actual and predicted hospital mortality were compared between patients with total PG < 0.420 mmol/l ("low PG") and patients with PG > or = 0.420 mmol/l. Mean total PG was 0.523 mmol/l, range 0.220-1.780 mmol/l. Low PG (n = 25) was associated with higher age (P = 0.03), shock as primary diagnosis, and higher actual hospital mortality (60 % vs 29 %, P = 0.01). Normal to high PG was associated with high plasma creatine phosphokinase (P = 0.007) There was a nonsignificant trend towards higher severity of illness scores and predicted mortality rates in the low PG group. The presence of low PG significantly improved mortality prediction when added as a factor to the APACHE II predicted mortality rate (P = 0.02). CONCLUSIONS: Low PG at acute ICU admission is related to higher age, shock as primary diagnosis, and higher hospital mortality. Low PG represents a risk of poor outcome, not fully reflected in the presently used mortality prediction systems.

Fasting and post-methionine homocysteine levels in NIDDM. Determinants and correlations with retinopathy, albuminuria, and cardiovascular disease.

OBJECTIVE: The increased cardiovascular risk in subjects with NIDDM is partly explained by an association with established risk factors like hypertension, dyslipidemia, and obesity. Mild hyperhomocysteinemia has emerged as a new risk factor for cardiovascular disease. The purpose of this study was to assess its role in NIDDM. RESEARCH DESIGN AND METHODS: We studied predictors of homocysteine levels and correlations between homocysteine and (micro-)albuminuria, retinopathy, and history of cardiovascular disease in normotensive NIDDM subjects under stable metabolic control. This was done in 85 NIDDM subjects by measuring fasting and post-methionine-loading homocysteine levels together with blood pressure, BMI, serum cholesterol, triglyceride, HDL cholesterol, folate, vitamin B12, pyridoxal-5-phosphate, HbA1c, and (micro-)albuminuria and creatinine clearance in triplicate 24-h urine samples. The relationship between micro- and macrovascular complications and fasting homocysteine only was studied in an additional 65 subjects, giving a total of 150 subjects. RESULTS: In multiple regression analysis, significant (P < 0.05) predictors of fasting homocysteine were low-normal values of creatinine clearance (threshold effect at < 80 ml.min-1 .1.73 m-2), folate (< 20 nmol/l), and vitamin B12 (< 350 pmol/l), and postmenopausal status in women. Determinants of post-methionine homocysteine were pyridoxal-5-phosphate levels < 80 nmol/l, creatinine clearance, and sex (higher levels in women). Hyperhomocysteinemia did not cluster with other cardiovascular risk factors, like hypertension, obesity, or dyslipidemia. Regarding cardiovascular complications, fasting homocysteine, but not post-methionine homocysteine, was higher in subjects with a history of cardiovascular disease. There was a stepwise increase in the prevalence of subjects with cardiovascular disease with increasing fasting homocysteine. The prevalence of cardiovascular disease was 19.4% in the bottom quartile of fasting homocysteine, versus 55.0% in the top quartile (P for trend < 0.01). Neither fasting homocysteine nor post-methionine homocysteine correlated with (micro-)albuminuria or with retinopathy. CONCLUSIONS: The findings suggest that homocysteine levels in NIDDM rise even with modest deterioration of renal function and when vitamin status is in the low to low-normal range. Fasting homocysteine correlates with macrovascular disease, but we found no evidence of a correlation with retinopathy or (micro-)albuminuria. Post-methionine homocysteine levels do not show a correlation with micro- or macrovascular complications.

Chemical analysis of an epidermoid cyst with unusual CT and MR characteristics.

Chemical analysis of the contents of a so-called bright epidermoid of the posterior fossa with unusual CT and MR imaging characteristics suggested that a combination of high protein content and high viscosity were responsible for the atypical imaging findings.

Multicentre evaluation of the EBIO plus glucose analyser.

The EBIO plus, a newly developed dedicated glucose analyser, was tested at three different sites for its analytical characteristics and practicability. Results were linear over a range of 2-50 mmol/l, precision was satisfactory (overall CV < 5.3% at a concentration level of 1.8 mmol/l and < 3.7% at a concentration level of 4.5-21 mmol/l). Calibration was stable up to 90 minutes. No significant influences were observed for several potential interfering substances at physiological or therapeutical levels. Practicability was found to be good. Sample through-put depends on calibration frequency and is maximally 166 per hour. Some problems with outliers had to be overcome in the beginning of the evaluation period, but in the end all evaluators concluded that the EBIO plus is user friendly with good analytical characteristics.

Marked interference of hyperglycemia in measurements of mean (red) cell volume by Technicon H analyzers.

Severe hyperglycemia can result in falsely high results for mean cell (erythrocyte) volume (MCV), which will also cause false results for erythrocyte indices calculated on the basis of MCV. Falsely high MCV results were obtained with the Technicon H1 and H2 analyzers and (to a lesser extent) with the Coulter T660. The H analyzers were more susceptible to this interference than was the Coulter T660. This difference in sensitivity of MCV to hyperglycemia can be explained by the use of sodium dodecyl sulfate in the Technicon erythrocyte diluent and by differences in incubation times. In severe hyperglycemia, results for MCV, mean cell hemoglobin concentration, and hematocrit obtained from electronic cell counters, especially Technicon H systems, are unreliable.

Effects of the modulating agent WR2721 on myelotoxicity and antitumour activity in carboplatin-treated mice.

The selective modulation of carboplatin [diammine(1,1-cyclo-butanedicarboxylato)platinum(II)]-induced myelotoxicity was investigated in mice, using the protective agent WR2721 [S-2-(3-aminopropylamino)ethyl-phosphorothioic acid, ethiofos]. In female BALB/c mice, WR2721 (200 mg/kg intraperitoneally, i.p.) partly prevented the reduction of in vitro proliferation of whole bone marrow cells and non-adherent cells when administered at different time points relative to 90 mg/kg carboplatin (i.p.). Protection was highest when WR2721 was administered 5 min prior to carboplatin. In vitro proliferation of whole bone marrow cells and non-adherent cells in liquid culture increased from 15% of control for carboplatin alone to 45% when WR2721 was administered 5 min prior to carboplatin. However, WR2721 did not significantly prevent the loss in clonogenic capacity of early hematopoietic progenitors in the bone marrow, as determined by a bilayered soft agar colony forming units assay. In nude mice, bearing well-established subcutaneous human ovarian carcinoma xenografts OVCAR-3, WR2721 (200 mg/kg i.p.) 5 min prior to intravenous carboplatin allowed a 1.5-fold increase in the maximum tolerated dose of carboplatin as determined by overall weight loss. WR2721 alone did not affect tumour growth. However, WR2721 had a potentiating effect on the tumour growth inhibition of a standard dose of carboplatin in this model. Minimal tumour volume compared to control (T/C) decreased from 9.4% with carboplatin alone to 2.2% with WR2721 5 min prior to the same dose of carboplatin. Specific growth delay (SGD) increased from 7.4 to 10.3. With the 1.5-fold increased, equitoxic dose of carboplatin in combination with WR2721, the antitumour activity was only slightly further increased (T/C = 1.4%, SGD = 10.5).

WR2721 as a modulator of cisplatin- and carboplatin-induced side effects in comparison with other chemoprotective agents: a molecular approach.

Cisplatin is an active cytostatic that became successful in the treatment of several types of solid tumours after its nephrotoxic potential was controlled by hydration and diuresis. Thiol compounds were tested to reduce further cisplatin-induced nephrotoxicity. Thiosulphate is rapidly excreted by the kidneys and protects against cisplatin-induced nephrotoxicity by inactivating reactive platinum species in the kidney. Due to inactivation of cisplatin in the circulation, thiosulphate also interferes with its antitumour activity. Therefore, it is mainly used in two-route schedules, whereby cisplatin is delivered locally to the tumour (i.p. or i.a.) while systemic (i.v.) thiosulphate protects the kidneys. Diethyldithiocarbamate was shown to protect against cisplatin-induced nephrotoxicity in several animal models by reversing cellular damage. However, in the clinic it has been less successful, partly due to its central nervous system toxicity. The endogenous thiol compounds glutathione and metallothionein have been shown to reduce cisplatin-induced toxicity both in animal models and in clinical trials. However, the results are rather preliminary and a reduction in therapeutic efficacy may be expected, for both glutathione and metallothionein have been reported to be involved in platinum resistance. The thioether methionine has been shown to reduce cisplatin-induced nephrotoxicity in animal models but it has not yet been tested in the clinic. Cisplatin-induced acute emesis can be sufficiently controlled with a new class of 5-hydroxytryptamine-3 (5HT3)-receptor blockers, but delayed emesis remains a problem. High-dose cisplatin regimens with protection of the kidneys induces ototoxicity, peripheral neuropathy and myelotoxicity, which become dose-limiting. Neurotoxicity was partly reversed by the neurogenerative agent ORG2766, but this agent does not reduce other cisplatin-induced toxicities. Therefore, an agent capable of protecting multiple non-tumour tissues is needed. Carboplatin is a second-generation analogue of cisplatin with less nephro-, neuro- and ototoxicity. Carboplatin is at least as active as cisplatin at its maximum tolerated dose, which is defined by its myelotoxicity. Protection from carboplatin-induced myelotoxicity may be controlled by autologous bone marrow transplantation and/or hematopoietic growth factor infusions. High-dose carboplatin schedules may cause nephrotoxicity, neurotoxicity and ototoxicity. Again, the protection of multiple non-tumour tissues is needed. WR2721 appears to be such a modulating agent capable of protecting multiple non-tumour tissues. It was shown to be preferentially metabolized and taken up as the thiol metabolite WR1065 by non-tumour tissues as compared with (hypoxic) solid tumours. It was shown to protect mice from cisplatin-induced nephrotoxicity and from cisplatin- and carboplatin-induced myelotoxicity without interfering with the antitumour activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of wr2721 (ethiofos) as modulator of Cisplatin-induced neurotoxicity studied at the ultrastructural level in the pond snail lymnaea-stagnalis.

Modulating effects of WR2721 were studied on cisplatin-induced histological and ultrastructural changes in the ganglia of the central nervous system of the pond snail Lymnaea stagnalis. The relevance of the study was indicated by showing histochemically that alkaline phosphatase, the enzyme converting WR2721 into the active drug WR1065, is present in the snail brain. Central nervous systems of the snail were either preincubated in 5 mM WR2721 or in snail Ringer for 1 h and then postincubated for 10 or 20 h in: (i) Ringer (control), (ii) WR2721 (5 mM), (iii) cisplatin (0.4 mM), or (iv) cisplatin (0.4 mM) plus WR2721 (5 mM). The following parameters were studied: (i) general morphology, (ii) chromatin (number and mean clump size per unit surface ama, clump size frequency distribution), (iii) nucleoli (ratio of granular/fibrillar areas, (iv), Golgi zones (mean surface area, area containing electron dense material), (v) secretory granules (number), (vi) lysosomes (number per unit surface area of cytoplasm). The focus was on two types of identified neuroendocrine cells. Incubation in Ringer alone (controls) caused slight inactivation of the cells between 10 and 20 h of incubation (e.g. relative decrease of the granular part of the nucleoli). WR2721 alone had comparable effects on the tissue. In addition, in this group, a reduction in the electron dense material of the Golgi zones was observed. No major differences in number of secretory granules were observed in any of the groups. Treatment with cisplatin alone for 20 h caused a disappearance of the orange colour of the ganglia, swelling of axons and distension of intercellular spaces. Such changes were not observed in the group treated with cisplatin plus WR2721 or any of the other experimental groups. Both cisplatin alone and cisplatin plus WR2721 caused an increase in the number of chromatin clumps and a decrease in the mean chromatin clump size after 10 and 20 h of incubation. With regard to these parameters no differences were observed between the two groups. The chromatin clump size distribution curves of both groups were significantly different from the curve of the controls. Compared to that of the cisplatin group, the curve of the cisplatin plus WR2721 group, especially after 10 h, had shifted in the direction of that of the controls. Treatment with cisplatin alone induced drastic changes in nucleolar morphology. After 20 h of incubation the nucleoli had transformed into homogeneous dense structures. After 10 h of incubation in cisplatin plus WR2721 nucleoli still had a normal appearance. After 20 h those of the co-treated group had also become electron dense and appeared to contain numerous dark dots. Cisplatin caused a significant decrease in the extent of the Golgi zones. Co-treatment with WR2721 prevented this decrease to a certain degree. In both groups the electron dense material had disappeared from the Golgi zones. Furthermore, cisplatin alone induced an increase in the number of lysosomes. This increase was slightly (but not significantly) prevented by co-treatment with WR2721. The observations on the snail neurons show that WR2721 at the concentration studied induced only minor morphological changes. The drug modulates to some extent the pathological changes caused by cisplatin. The results support clinical data indicating that cisplatin-induced neurotoxicity is reduced by WR2721.

Cytostatic activity of cisplatin in the presence of WR2721 and its thiol metabolite WR1065 in OVCAR-3 human ovarian cancer cells as compared to V79 fibroblasts.

The effect of the modulating agent WR2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid, ethiofos) and its active thiol metabolite WR1065 on the cytostatic effect of cisplatin was investigated in a human ovarian cancer cell line OVCAR-3 and compared to that in V79 fibroblasts. WR1065 protected both OVCAR-3 and V79 cells against the cytostatic effect of cisplatin when incubated together with (co-incubation) or 15 min prior to (pre-incubation) the platinum drug. A dose-related effect up to complete protection was obtained when cells were coincubated with WR1065. Pre-incubation with WR1065 protected less and protection declined as cisplatin exposure time increased. A 60 min incubation of the cells with WR1065, starting 1 hr after a 60 min cisplatin exposure, did not protect OVCAR-3 and V79 cells from the cytostatic effect of cisplatin. The parent drug WR2721, up to a concentration of 0.01 M, did not protect OVCAR-3 and V79 cells when co-incubated with cisplatin. These results support the conclusions: a) that WR1065, and not the parent drug WR2721, is the protective species entering the cells, b) that prevention of damage is the main mechanism of protection and c) that tissues are expected to be protected by WR2721 only if WR1065 is formed and taken up by these tissues. Therefore, the preferential formation and uptake of WR1065 by non-tumour tissues is essential for a successful combination of WR2721 with platinum chemotherapy.

Time dependence of the selective modulation of cisplatin-induced nephrotoxicity by WR2721 in the mouse.

2-(3-Aminopropylamino)ethylphosphorothioic acid (WR2721; ethiofos) was shown to selectively protect nontumor tissues from cis-diamminedichloroplatinum(II) (cisplatin)-induced toxicity, when administered 30 min prior to the platinum drug. Selectivity of protection by WR2721 is probably due to the preferential formation and uptake of the thiol metabolite 2-(3-aminopropylamino)ethanethiol (WR1065), which can inactivate toxic platinum-species inside the cell. We investigated the protective potential of WR2721, when administered at different time points relative to cisplatin. BALB/c mice treated with WR2721 (200 mg/kg i.p.) either 30 min or 5 min prior to cisplatin (i.p.) allowed a 2.2-fold increase in cisplatin dose to 19 mg/kg before the occurrence of nephrotoxicity as expressed by an increase in plasma urea. A small part of the protection could be ascribed to the mannitol (200 mg/kg), present in the formulated WR2721. WR2721 (200 mg/kg) 30 min after 14.5-16-mg/kg cisplatin did not offer any protection against the rise in plasma urea. WR2721 (200 mg/kg) 5 min before 19-mg/kg cisplatin did not cause liver toxicity (increase in serum glutamic pyruvic transaminase or serum glutamic oxaloacetic transaminase). Furthermore, WR2721 (200 mg/kg) 5 min prior to cisplatin did not reduce antitumor activity in nude mice bearing well-established human ovarian cancer xenografts. Under protection of WR2721, the dose of cisplatin could be increased by a factor of 1.6 to 8 mg/kg (administered twice weekly), resulting in an increased antitumor activity.

Effects of the modulating agent WR2721 and its main metabolites on the formation and stability of cisplatin-DNA adducts in vitro in comparison to the effects of thiosulphate and diethyldithiocarbamate.

The influence of the modulating agent WR2721, its active thiol-metabolite WR1065 and the symmetrical disulphide WR33278 on the in vitro formation and stability of cis-diamminedichloroplatinum(II) (cisplatin, CDDP)-DNA adducts was investigated and compared with the effects of the highly nucleophilic modulating agents diethyldithiocarbamate (DDTC) and thiosulphate (TS). Salmon sperm DNA (0.5 mg/mL) was incubated with 25 micrograms/mL (83 microM) cisplatin for 1 hr in 50 mM phosphate buffer, pH 7.2 at 37 degrees in the absence or presence of modulating agent. DDTC and TS were potent inhibitors of the platination of the DNA (95 and 89%, respectively, with 4.2 mM of modulating agent). The WR-compounds were also remarkably active in the inhibition of DNA platination. Prevention of adduct formation in the presence of 4.2 mM WR-compound decreased in the order WR1065 (74%) greater than WR33278 (63%) greater than WR2721 (51%). The prevention of CDDP-DNA adduct formation by WR1065 was strongly concentration-dependent up to 4.2 mM but at higher concentrations this protection hardly increased at all. In the presence of the modulating agents, increased levels of CDDP monofunctionally bound to a guanine residue were observed with a simultaneous decrease in the relative abundance of bifunctional adducts. All modulators were also able to reverse part of the CDDP-DNA adducts formed. After a 2-hr incubation of already platinated salmon sperm DNA with 4.2 mM of modulating agent, the removal of Pt from DNA amounted to about 43% with DDTC, 28% with WR1065 and 13-14% with TS, WR2721 and WR33278. Even CDDP bifunctionally bound to two adjacent guanines in the same DNA strand, which is considered to be a very stable adduct, was partly reversed. Our observations suggest that WR2721, especially when administered prior to or concomitantly with CDDP, can be expected to protect those tissues from CDDP-induced damage to DNA that are able to efficiently dephosphorylate WR2721 followed by uptake of the thiol metabolite WR1065. This stresses the importance of a selective formation and uptake of WR1065 by non-tumour tissues for the successful use of WR2721 as a protective agent in combination with platinum-based cancer chemotherapy.

Effect of WR-2721 on the toxicity and antitumor activity of the combination of carboplatin and 5-fluorouracil.

We evaluated the effects of WR-2721 on the toxicity and antitumor activity of the combination of 5-fluorouracil (5FU) and carboplatin (CBDCA) in BALB/c and C57B1/6 mice. On a weekly schedule, i.p. injection of 200 mg/kg WR-2721 at 5 min prior to the administration of this combination enabled us to increase the CBDCA dose from a nontoxic level of 45 mg/kg to a normally toxic dose of 60 mg/kg in non-tumor-bearing BALB/c mice while maintaining the 5FU dose at 100 mg/kg. When WR-2721 was given 30 min before this combination, the CBDCA dose could not be increased to 60 mg/kg without producing drug-related deaths. WR-2721 protected against CBDCA- and 5-FU-induced thrombocytopenia but did not prevent leukopenia or anemia in C57B1/6 mice. The antitumor activity of the combination against colon 26 tumors in BALB/c mice was increased by pretreatment with WR-2721, which facilitated elevation of the CBDCA dose to 60 mg/kg in combination with 100 mg/kg 5FU. These results reveal better therapeutic efficacy for the combination of 5FU and CBDCA following pretreatment with WR-2721.

The reversal of cisplatin-protein interactions by the modulating agent WR2721 and its metabolites WR1065 and WR33278.

The reversibility of cisplatin-protein interactions by the modulating agent WR2721, its active thiol-metabolite WR1065, and the symmetrical disulfide WR33278 was studied using the model compounds (Pt(diethylenetriammine) monofunctionally bound to the sulfur in glutathione (Pt(dien)SG) and Pt(diethylenetriammine) monofunctionally bound to the sulfur in S-methylglutathione (Pt(dien)SMeG). Both model compounds could be quantified by high-performance liquid chromatography (HPLC) with UV detection. The Pt-cysteine-like bond in Pt(dien)SG could not be reversed by any of the WR compounds or by the strong nucleophiles thiosulfate (TS) and diethyldithiocarbamate (DDTC). However, the Pt-methionine-like bond in Pt(dien)SMeG could be reversed by WR1065, although the reversal was slow (k2 = 0.142 M-1 s-1) as compared with that obtained using the modulating agents TS (k2 = 10.1 M-1 s-1) and DDTC (k2 = 3.66 M-1 s-1). WR2721 was hardly able to reverse the Pt-S bond in Pt(dien)SMeG (k2 = 0.00529 M-1 s-1), and WR33278 showed no capacity to do so. The activity of cis-diamminedichloroplatinum(II) (CDDP)-inactivated fumarase was not appreciably restored by any of the WR compounds (16%, 7.7%, and 0 for 20 mM WR1065, WR2721, and WR33278, respectively) in contrast to the strong nucleophile DDTC (61% for 2 mM DDTC). These in vitro studies provide information at the molecular level that may explain why WR2721, in contrast to DDTC, does not provide protection against cisplatin-induced nephrotoxicity when it is given after platinum-containing chemotherapy. The results support the present clinical use of WR2721 prior to the administration of platinum compounds.

Protection by WR-2721 of the toxicity induced by the combination of cisplatin and 5-fluorouracil.

We evaluated the effects of WR-2721 and its metabolite WR-1065 on in vitro growth inhibition by 5-fluorouracil (5FU) and cisplatin (CDDP) and the effect of WR-2721 on in vivo toxicity and antitumor effect of 5FU and CDDP. In cell culture both WR-2721 and WR-1065 were not able to reverse growth inhibition caused by either 5FU or CDDP. Administration of WR-2721 i.p. at 525 mg/kg to mice resulted in a severe temperature drop to 27 degrees C; at 200 mg/kg hypothermia was less severe. WR-2721 failed to prevent 5FU toxicity, but the maximum tolerated dose of CDDP in the combination with 5FU (at 100 mg/kg) could be increased from 3 to 7 mg/kg. CDDP at 7 mg/kg enhanced leukopenia caused by 5FU at 100 mg/kg to 20% and thrombocytopenia to 40%; WR-2721 reduced leukopenia and prevented thrombocytopenia induced by the combination. Combination of CDDP, 5FU, and WR-2721 resulted in an enhanced antitumor activity against the murine colon tumor Colon 26 compared to 5FU alone and to 5FU combined with CDDP at their maximum tolerated dose.

Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.

Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.

The chemical reactivity of the modulating agent WR2721 (ethiofos) and its main metabolites with the antitumor agents cisplatin and carboplatin.

The antitumor agents cisplatin [cis-diamminedichloroplatinum(II), CDDP] and carboplatin [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), CBDCA] can react with a nucleophilic agent by a direct ligand exchange of the (labile) anionic ligands or through hydrolysis of these ligands followed by a fast reaction of the hydration product with the nucleophile. At pH 7.4 and 37 degrees, CDDP and CBDCA were incubated with several molar excesses of the modulating agent WR2721, its active thiol metabolite WR1065 or the symmetrical disulphide WR33278. The reaction rate constants for the hydrolysis and the direct inactivation by the WR-compounds were obtained from the pseudo first-order disappearance of the intact Pt-drug, with or without the WR-compounds at molar ratios of 50, 100 and 200. The hydrolysis of carboplatin (kaq,CBDCA = 2 x 10(-8) M-1 sec-1) was 100-fold less rapid than that of cisplatin (kaq,CDDP = 2 x 10(-6) M-1 sec-1). However, direct inactivation by WR2721, WR1065 and WR33278 was only 4-, 4- and 22-fold less rapid for carboplatin than for cisplatin, respectively. This direct inactivation was slow compared to the strong nucleophiles thiosulphate (TS) and diethyldithiocarbamate (DDTC) and decreased for both Pt-drugs in the following order: WR1065 (kWR1065/CDDP = 49.1 x 10(-4) M-1 sec-1, kWR1065/CBDCA = 12.4 x 10(-4) M-1 sec-1) greater than WR2721 (kWR2721/CDDP = 25.3 x 10(-4) M-1 sec-1, kWR2721/CBDCA = 6.07 x 10(-4) M-1 sec-1) greater than WR33278 (kWR33278/CDDP = 8.60 x 10(-4) M-1 sec-1, kWR33278/CBDCA = 0.39 x 10(-4) M-1 sec-1. Thus for CDDP, the hydrolysis-mediated interaction with the WR-compounds contributed more to the disappearance of intact platinum antitumor agent than it did for CBDCA. Considering the relatively low reactivity of WR2721 and its main metabolites with the platinum antitumor agents, in addition to their pharmacokinetic behavior, a significant inactivation of the platinum antitumor drugs by WR2721 and its main metabolites is, in contrast to TS, not expected in the circulation.

Comparative toxicokinetics of 2,2'- and 4,4'-dichlorobiphenyls in the pond snail Lymnaea stagnalis (L.).

Pond snails (Lymnaea stagnalis (L.)) were treated with 2,2'-dichlorobiphenyl (DCB) or 4,4'-DCB, to examine the toxicokinetic profile of these compounds. Snails were treated orally with 210 micrograms 4,4'-DCB (impregnated on food) for 14 hr, or snails were injected with 50 micrograms of 2,2'-DCB or 4,4'-DCB in the foot. At different times after starting feeding or injection, tissues (albumen gland, digestive gland and digestive tube, central nervous system, remainder parts), hemolymph and faeces were analyzed for unchanged 2,2'- or 4,4'-DCB. The results showed that in case of oral administration of 4,4'-DCB after 144 hr, 97.5% of the dose was excreted unchanged in the faeces. During the first 48 hr 4,4'-DCB was found in all tissues. Thereafter, an exponential elimination was found (the rate constant of elimination (kel) varied from 0.010-0.021 per hr, t1/2 from 33-60 hr and the apparent clearance from 0.02-0.3 g/hr for the different tissues). After injection, the compounds were found in all the above mentioned tissues, especially in the digestive gland. There was a clear difference between snails injected with 2,2'- and 4,4'-DCB. Firstly, kel for 2,2'-DCB was higher (0.028 per hr vs 4,4'-DCB: 0.001 per hr). Secondly, 2,2'-DCB was lethal; 63% of the animals died after 72 hr.

Determination of pentachlorophenol in wood samples using liquid chromatography with UV absorbance, amperometric and electron-capture detection.

The potential of reversed-phase liquid chromatography (RPLC) with UV and amperometric detection (AD), and of normal-phase liquid chromatography (NPLC) with UV and electron-capture detection (ECD) for the determination of pentachlorophenol (PCP) in wood samples has been studied. When PCP concentrations of at least 1-5 ppm have to be determined, RPLC-UV and RPLC-AD on C18-modified silica are useful techniques, provided a two- or three-step sample-preparation step is used. NPLC-UV on bare silica columns does not offer any advantage over RPLC-UV. NPLC-ECD on bare silica and with an acidified toluene-hexane mixture as eluent offers good selectivity and sensitivity, as well as satisfactory linearity and reproducibility for the determination of PCP in wood samples down to low ppb levels. Use of the two-step clean-up procedure is sufficient, and even a single-step procedure has been utilized. In the latter case, analysis times are longer because of the presence of late-eluting ECD-active interferences. The two-step clean-up procedure generally used involves a liquid-liquid extraction with dichloromethane, and solid-liquid sorption using a Sep Pak C18 cartridge. PCP recovery over the 0.2-10 ppm range is 75-100%. Several wood samples containing 1-50 ppm of PCP have successfully been analyzed, and the good potential of NPLC-ECD for trace-level determination of PCP has been demonstrated.