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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat and fish, is carcinogenic to the mammary glands of rats. Mutations in the H-ras gene were here examined in PhIP-induced mammary tumors of female F344 rats by the polymerase chain reaction followed by single strand conformation polymorphism analysis (PCR-SSCP) and restriction enzyme length polymorphism analysis (RFLP). Mutations in codon 12 of the H-ras gene were detected in four out of 13 tumors by PCR-SSCP. Three of them were GGA to GAA, and one was GGA to GTA. However, by RFLP analysis, four additional mutations in codon 13 were also detected in the same samples. Two had a GGC to CGC mutation, and the other shifts were GGC to GAC and GGC to GTC. Therefore, overall eight out of 13 cases had H-ras gene mutations. These results indicate that changes in H-ras function may play important roles in PhIP-induced mammary carcinogenesis.

We demonstrated the dissociation between plasma adrenocorticotropin (ACTH) and serum cortisol levels during the early recovery period after radical gastrectomy in 9 of 31 patients with gastric adenocarcinoma. Patients with the dissociation between plasma ACTH and serum cortisol levels (DAC) showed a sustained elevation of serum cortisol level on the first or second postoperative day, while the plasma ACTH level returned to its preoperative state. These patients also had more advanced cancers (p < 0.05) and suffered from more postoperative complications (p < 0.05) than those without DAC. In these patients with DAC, serum cortisol and interleukin (IL)-6 levels remained higher on the second postoperative day than in those of the patients without DAC (21.80 +/- 1.57 vs. 13.68 +/- 0.72 microg/dl, p < 0.001, and 74.31 +/- 15.65 vs. 18.75 +/- 3.14 pg/ml, p < 0.001, respectively). On the second postoperative day, serum IL-6 levels showed a significant correlation with serum cortisol levels in all patients (r = 0.511, p < 0.01). These results suggest that the DAC during the early postoperative period after radical gastrectomy is associated with advanced stage of cancer and postoperative complication, and that the increased serum IL-6 level is at least in part responsible for maintaining the elevated serum cortisol., (Copyright 2000 S. Karger AG, Basel)

Anophthalmia in litters of pregnant rats treated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a food-derived carcinogen, was incidentally encountered in a risk-assessment study with 2-generation exposure to PhIP. Female Fischer 344 animals were given 200 ppm PhIP in the diet for 4 wk before mating with nontreated males and also during gestation and lactation periods. Mean numbers of newborn rats per litter in control and PhIP-treated groups were 7.9 +/- 2.9 and 7.1 +/- 1.6 in trial 1 and 8.3 +/- 1.9 and 6.1 +/- 2.4 in trial 2. Among 49 (trial 1) and 63 (trial 2) offspring from PhIP-treated dams, 9 (18.4%) and 32 (50.8%) demonstrated anophthalmia, and 1 (2.0%) and 8 (12.7%) demonstrated hydrocephaly. Five of 7 (71.4%) and 13 of 14 (92.9%) dams delivered pups with malformations in trials 1 and 2, respectively. Also, in a previous study that was carried out with the same protocol and that used the Sprague-Dawley strain of rats, anophthalmia and hydrocephaly were observed in 2 and 1 out of 175 pups, respectively, from 100 ppm PhIP-treated dams. No congenital malformations were found in control groups of the same size in either experiment. In addition to having been previously identified as a cause of carcinogenic activity, our findings suggest that PhIP is capable of causing anophthalmia in rats when administered during the gestational period.

To explore the role of ester groups in the neuromuscular blocking (NMB) property of various quaternary ammonium tropinesters, we have synthesized and pharmacologically evaluated twenty bisquaternary ammonium derivatives where two tropane (I), 3alpha-hydroxytropane (II), or 3-oxotropane (III) moieties were connected on their nitrogen atoms with a polymethylene chain of C3-C12 length. The rank order of potency of these agents in the rat (evoked EMG responses of the ant. tibial muscle) was: I, II, and III. Most potent were the longer chain (e.g. C10-C12) compounds of series I, yet they were inferior to bisquaternary ammonium tropinester derivatives (e.g. N,N'-polymethylene bisquaternary tropinesters, or bisquaternary derivatives of dicarboxylic acid tropinesters). Although some of the agents (series I and II) showed a rapid onset and short duration of action-profile, they also exhibited cardiovascular side effects (e.g. cardiac vagal block and blood pressure changes). In the design of new NMB agents of rapid onset and short duration of action the presence of different ester groups in addition to an azabicyclo ring system (e.g. tropane), seems to be essential.

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Mazindol, 5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol, has been shown to be an effective anorexic. To explore the structure-activity relationships, several 1-ethyl-3-substituted-4-aryl-4-hydroxyindeno[1,2-c]pyrazoles were prepared and subjected to various animal screens. The 1-ethyl-3-tert-butyl-4-aryl-4-hydroxyindeno[1,2-c]pyrazoles are capable of significantly depressing forced and spontaneous motor activity in mice but have low LD50's. Two of these compounds were tested in an Ehrlich ascites tumor screen. The 1-ethyl-3-phenyl-4-aryl-4-hydroxyindeno[1,2-c]pyrazoles depressed forced and spontaneous motor activity at low doses and were relatively nontoxic.

Six pyridine-2-carboxaldehyde, one pyridine N-oxide 2-carboxaldehyde, and five diketone thiophosphoric hydrazones, three thiophosphoric hydrazides, and two cupric chelates were synthesized. The chelates and nine of the hydrazones were tested against Ehrlich ascites carcinoma. Seven of these latter agents were administered concurrently with either cupric and/or ferrous salts to mice bearing this tumor. The greatest activity was found with the chelate, cimethyl pyridine-2-carboxyaldehyde phosphorothioic hydrazone-copper (1:1). The hydrazone portion of this chelate also formed a ligand-copper (2:1) complex. Although all of the hydrazones but one were inactive when evaluated alone, the concurrent injection of cupric ion increased survival times by an avoli alkaline phosphatase was found to be inhibited by two thiosemicarbazones in a manner similar to that previously reported by these agents against alkaline phosphatase derived from Sarcoma 180-6-thiopurine resistant ascites cells. None of the 14 hydrazides or hydrazones tested against E. coli enzyme displayed significant inhibition.