Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride.

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

Organotin-protein interactions. Binding of triethyltin bromide to cat haemoglobin.

Triethyltin binds to native cat and rat haemoglobin but not to their denatured forms or to other animal haemoglobins. Two molecules of the organotin bind to one molecule of R-state cat haemoglobin with affinity constants of about 1 X 10(5) M-1. Little or no triethyltin is bound to the deoxygenated (T-state) protein. Binding appears to be dependent upon the existence of a specific three-dimensional configuration of cysteine and histidine residues. The properties of the triethyltin-cat haemoglobin complex are consistent with those of a haemoglobin conformer whose allosteric equilibrium is displaced toward the R-state. Its oxygen affinity and rate of oxidation by nitrite is increased, and the rate of reduction of the methaemoglobin derivative by ascorbate is decreased. These effects of triethyltin are opposite and antagonistic to the effects of inositol hexaphosphate. They are exerted on the alpha- as well as beta-haem groups, even though triethyltin is bound at sites on alpha-globin far removed from the haem groups.

Tin distribution in adult rat tissues after exposure to trimethyltin and triethyltin.

The time course of distribution of tin in the adult rat was determined in brain, liver, kidneys, heart, and blood following single ip administrations of trimethyltin hydroxide (TMT) and triethyltin bromide (TET). Adult Long-Evans rats were killed 1, 4, 12, and 24 hr, and at 5, 10, or 22 days following injection of TMT and TET (N = 6/time), and tissues were analyzed for total tin by atomic absorbance spectroscopy. TET exposure resulted in higher tin concentrations in brain, liver, and kidney tissues, while the two trialkyltins resulted in approximately equal tin concentrations in the heart and blood. Rates of elimination of tin (expressed as elimination rate constants, Kel) were greater in all tissues following TET exposure than following TMT exposure. The concentration of tin in the brain 12 hr after TMT exposure was 4.4, 8.5, and 12.7 ng tin/mg protein for dosages of 3.0, 6.0, and 9.0 mg/kg, respectively. Tin was evenly distributed across the cerebellum, medulla-pons, hypothalamus, hippocampus, and striatum following TMT exposure. These results describe major differences in the disposition and rates of elimination of tin from body tissues after TMT and TET exposure, and demonstrate that the regional disposition of tin is not related to the region-specific pathology reported following TMT exposure.

Distribution of tin in brain subcellular fractions following the administration of trimethyl tin and triethyl tin to the rat.

The time course of tin distribution in homogenates and subcellular fractions of rat brain was determined following the acute administration of trimethyl tin (TMT) and triethyl tin (TET) to the rat. Exposure to TMT resulted in lower concentrations but greater persistence of tin in subcellular fractions compared to exposure to TET. A delayed accumulation of tin in the mitochondrial fraction was observed following the administration of TMT but not TET. Analysis of total protein and mitochondrial markers did not reveal differences between the compositions of mitochondrial fractions prepared from control and TMT-treated subjects.

Tin distribution in adult and neonatal rat brain following exposure to triethyltin.

The uptake, distribution, and elimination of tin were determined in adult and neonatal (Postnatal Day 5) rat brain following ip administration of triethyltin bromide (TET). Groups of five adult CD rats were killed at 10 min, 1 hr, 4 hr, 24 hr, 5 days, or 10 days following acute exposure to 6.0 mg/kg TET; an additional group of adult animals was killed at 24 hr following exposure to either 3.0, 6.0, or 9.0 mg/kg (N = 5/dosage). The time course for tin distribution in 5-day-old rat pups was determined by killing pups 10 min, 30 min, 1 hr, 4 hr, 8 hr, 12 hr, 24 hr, 5 days, 10 days, or 22 days following exposure to either 3.0 or 6.0 mg/kg TET (N = 4/dosage/time). Tin analyses were performed by flameless atomic absorption spectrophotometry. The t1/2 for total tin in the adult rat brain following 6.0 mg/kg TET was determined to be 8.0 days. The maximum concentration in the adult was reached at 24 hr and corresponded to 4.6, 9.6, and 16.6 ng tin/mg protein for dosages of 3.0, 6.0, and 9.0 mg/kg, respectively. Tin was evenly distributed across all brain areas studied. For animals exposed to 6.0 mg/kg TET on Postnatal Day 5, the t1/2 for total tin in the brain was 7.3 days. A maximum concentration of 9.9 ng tin/mg protein was reached at 8 hr postexposure. The rate of elimination of tin from the brain (as measured by the elimination rate constant kel) did not differ significantly between adults and neonates. However, due to a dilution effect by the rapid brain growth of the neonate, the concentration of tin in the neonatal brain following TET administration decreased significantly faster than that in the adult.

The microsomal metabolism of the organometallic derivatives of the group-IV elements, germanium, tin and lead.

The NADPH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetra-ethyl-substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product. These reactions were shown to be catalysed by the liver microsomal cytochrome P-450-dependent mono-oxygenase. Since formation of ethane and ethylene was differentially inhibited by anaerobiosis, the results suggest that at least a large portion of the ethane produced may be derived by a reductive mechanism. Triethyltin bromide in both the absence and presence of NADPH was shown to convert cytochrome P-450 into cytochrome P-420 and to affect the function of the mono-oxygenase in vitro. Tetraethyltin caused the NADPH- and time-dependent formation of cytochrome P-420, suggesting that tetraethyltin is converted into triethyltin salts in significant concentrations. The order of potency in formation of cytochrome P-420 was closely paralleled by the ability of the tin derivatives to induce microsomal lipid peroxidation in vitro.

The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies.

Purified glutathione(GSH)-S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEt3). With 1-chloro-2,4-dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10(-3) M the inhibition constants for transferases A and C at 25 degrees C are similar and very low, 3.2 . 10(-8) M and 5.6 . 10(-8) M respectively, whereas for transferase B the inhibition constant is 3.5 . 10(-5) M. Equilibrium-dialysis experiments carried out at 4 degrees C in the absence of GSH give apparent dissociation constants of 7.1 . 10(-4) M and 3.4 . 10(-4) M for transferases A and B respectively, but if 5 . 10(-3) M glutathione is included in the dialysis solutions these values fall to 2.0 . 10(-7) M and 2.6 . 10(-5) M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSH-SnEt3, in which GSH and SnEt3 may or may not interact directly; or are possibly quaternary complexes, enzyme-(GSH)2-SnEt3. SnEt3 could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases.

Triethyltin binding to cat haemoglobin. Evidence for two chemically distinct sites and a role for both histidine and cysteine residues.

Triethyltin binding to cat haemoglobin was measured after pretreatment of the protein with diethyl pyrocarbonate at pH 6.0,iodoacetamide or phenylmercuric acetate or by photo-oxidation in the presence of Methylene Blue. The pentaco-ordinate nature of the binding of triethyltin to cat haemoglobin is confirmed by the inability of intramolecularly pentaco-ordinate tin compounds to compete. Consideration of the symmetry of the haemoglobin molecule in the light of the above results suggests that a unique arrangement of histidine and cysteine residues is required for the binding of triethyltin. The effects of treatment with diethyl pyrocarbonate of other preparations which bind triethyltin (rat liver supernatant, a fraction from rat liver mitochondria and rat brain myelin) were determined and shown to be complex.

Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate.

Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.