Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins.

The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

High-throughput synthesis and screening of platinum drug candidates.

Platinum drugs play an important role in the treatment of cancer, but there is room for improvement. Here we present a new platinum drug-discovery strategy to identify compounds having efficacy equivalent to that of cisplatin with the expectation that some may increase the spectrum of treatable tumors and/or reduce dose-limiting toxicity. Platinum drug candidates were generated through the use of automated synthesis, taking advantage either of the trans effect or by using silver chloride precipitation to activate the starting materials. Reaction products were screened for activity in a high-throughput transcription assay and the most promising candidates characterized. Over 3,600 reaction products were screened for their ability to inhibit transcription of beta-lactamase in the BlaM HeLa cell line by monitoring cleavage of a lactam ring linking the two halves of a fluorescent resonance energy transfer (FRET) dye, CCF2/AM. From this screen, three reactions produced good candidates, and four species were identified among these reaction products. Three of the compounds, cis-[(isopropylamine)2PtCl2], cis-[(cyclobutylamine)2PtCl2], and cis-[ammine(cyclobutylamine)PtCl2], have been previously determined to be active cisplatin analogs. The fourth compound, cis-[ammine(2-amino-3-picoline)PtCl2], represents a new kind of antitumor drug candidate similar to ZD0473, a recently reported analog. The discovery of these compounds represents an important proof of principle that platinum anticancer drug candidates can be rapidly prepared and screened in this manner.

An ethologist's approach to behavioural toxicology.

Ethology, the biology of behaviour, is briefly introduced, and a review is presented of two ethological methods applied to toxicology. (1) When elements in the behaviour of rats in a social situation are objectively observed, chemically-induced changes in several kinds of behaviour can be detected at low doses. Changes are specific and repeatable in long-term experiments. Effects of nicotine at a 'smoking' dose, or trichloroethylene at the Threshold Limit Value, were reversible; limited tolerance developed to methyl mercury dicyandiamide. (2) An 'Exploration-Thirst' method is simple enough for use as a screen. It was compared with conventional toxicological methods for 30 compounds in routine screening (20 acute intraperitoneal, 10 subacute inhalation). Both ethological methods are sensitive enough to estimate 'no-effect' doses. They also distinguish non-specific toxic effects (consistent with the animal's equivalent of 'feeling ill') from various more specific CNS effects comparable to those of human experience.

General toxicology of tin and its organic compounds.

The toxicology of tin is almost entirely the toxicology of the organic compounds of tin, for the metal itself and its inorganic compounds appear to be nearly harmless for practical purposes. Furthermore, the neurotoxicity of organotin is essentially that of trimethyltin and triethyltin.

Early change and adaptation in the social behaviour or rats given methyl mercury in the diet.

Rats were fed on diets containing methyl mercury dicyandiamide (MMD) at concentrations of 1.5, 7.5 or 75 ppm, and observations made of their social and exploratory behaviour and of gross neurotoxicity (ataxia). Mercury concentration in the blood was monitored. MMD at 75 ppm (50 ppm Hg) for 24 days caused ataxia with a sudden onset at 21-27 days. Social behaviour was reduced at 16-17 days. In two experiments at 7.5 ppm MMD, activity was increased in observations of social behaviour after 2 to 17 days, and treated rats found water in an unfamiliar cage sooner than controls. No difference from controls was apparent from then until increased activity re-appeared after 9 mon of the diet (exp. 1) or after 7 days recovery from 31 or 45 days diet (exp. 2). Ataxia was not observed after 7.5 ppm MMD for up to 47 weeks or in 14 weeks recovery. No consistent effect was observed at 1.5 ppm MMD for 31-45 days. MMD, therefore, had a behavioural effect in rats, independent of gross neurotoxicity; its details were consistent with a hyperresponsiveness to stimuli. Tolerance occurred to this effect at a time when blood-mercury concentration was still rising, but the tolerance appeared to be subject to overload.

Behaviour of rats exposed to trichloroethylene vapour.

Rats were exposed to trichloroethylene (TCE) vapour for about five five-day weeks at concentrations from 100 to 1 000 ppm, and at 100 ppm for 12 1/2 weeks. The social behaviour of paired male rats was observed periodically in the home cage for five minutes after they had been exposed to TCE. The principal finding was a consistent reduction of up to 24% in the total acitivity. A single day's exposure to TCE was sufficient at the highest concentration. At 100 ppm, a similar decline in activity was significant after 1 1/2 weeks' exposure in one experiment and 8 1/2 weeks' in another. The decline in activity was fairly uniform and not usually because of specific reductions in particular kinds of behaviour. However, exploration of the cage and submission to, or escape from, the other rat were sometimes specifically reduced. In an 'exploration-thirst' test, rats were deprived of water overnight and placed on two or three occasions in a previously unfamiliar cage. Rats exposed to 100, 200, or 1 000 ppm TCE found water and began drinking sooner than their controls without altering the rate of movement about the cage. These results suggest lowered performance in a familiar situation where rats are usually very active and some loss of inhibitory control in an unfamiliar one. At the present threshold limit value, repeated exposure to TCE eventually had effects similar to those of one day's exposure to a higher concentration, but only after a widely variable delay.