Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome p450 enzymes.

Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHITC), and sulforaphane (SFN), expressed as the first-order rate constant k(1) and the half-life of decomposition Dt(1/2), were measured in aqueous solutions at pH 7.4 and 37 degrees. The Dt(1/2)s for the Cys conjugates were severalfold shorter than the Dt(1/2)s for respective GSH conjugates, while the Dt(1/2)s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 microM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.

[Small bowel primary neoplasm as a cause of jejunal invagination and mechanical ileus]. / Pierwotny nowotwór zlosliwy jelita cienkiego przyczyna wglobienia jelita czczego i niedroznosci przewodu pokarmowego.

Intestinal invagination causing intermittent ileus has been described. The invagination was cause by jejunal primary neoplasm. Difficulty with diagnosis and oncological operation necessity in every case of intestinal tumor has been emphasise.

Synthesis of anti-1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-6-nitrochrysene and its reaction with 2'-deoxyguanosine- 5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, and calf thymus DNA in vitro.

The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models, and its environmental presence, suggest its potential importance with regard to human cancer development. Depending on the bioassay model, 6-NC can be activated by simple nitro reduction, ring oxidation, or by a combination of ring oxidation and nitro reduction. Only the first pathway has been clearly established. Thus, this study purports to unequivocally define the other pathways. Toward this end, we report for the first time the synthesis of anti-1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-6-nitrochrysene (6-NCDE), a likely ultimate carcinogenic metabolite of 6-NC. Also, we describe our initial investigation of its binding with calf thymus DNA, 2'-deoxyguanosine-5'-monophosphate (2'-dGuo), and 2'-deoxyadenosine-5'-monophosphate (2'-dAdo) in vitro. These adduct markers were then employed for comparison with those obtained in the rat after in vivo treatment with 6-NC. On the basis of the results, it appears that the major adduct formed in the liver of rats treated with 6-NC is not derived from 6-NCDE.

Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate.

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.

["Putting on a stake"-type of trauma as a cause of multiple injuries in abdominal and thoracic cavities]. / Uraz typu "wbicie na pal" przyczyna mnogich obrazen narzadów jamy brzusznej i klatki piersiowej.

"Pale sink" trauma in 16 years old boy has been described as a cause of multi-organ injuries.

Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky benzo[a]pyrene diol epoxide-deoxyguanosyl adducts.

Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.

Specificities of human glutathione S-transferase isozymes toward anti-diol epoxides of methylchrysenes.

The specificities of human glutathione (GSH) S-transferase (GST) isozymes of class alpha (hGSTA1-1), mu (hGSTM1-1) and pi (hGSTP1-1), including the three allelic forms of hGSTP1-1 [hGSTP1-1(I104,A113), hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113)], in catalyzing the GSH conjugation of anti-diol epoxide stereoisomers of 5-methylchrysene (anti-5-MeCDE) have been examined. The specific activities of human GSTs were significantly higher toward (+)-anti-5-MeCDE than toward the (-)-enantiomer of anti-5-MeCDE. All three variants of hGSTP1-1 were significantly more efficient than either hGSTA1-1 or hGSTM1-1 in GSH conjugation of (+)-anti-5-MeCDE. The catalytic efficiencies of hGSTP1-1 variants toward (+)-anti-5-MeCDE were in the order hGSTP1-1(I104,A113) > hGSTP1-1(V104,V113) > hGSTP1-1(V104,A113). The present study suggests that the I104,A113 allele, which is most frequent in human populations, may play a major role in the detoxification of (+)-anti-5-MeCDE. This may point to specificity, because previous studies from our laboratory have shown that the hGSTP1-1(V104,V113) isoform is significantly more efficient than the other two variants of hGSTP1-1 in catalyzing GSH conjugation of (+)-anti-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], the ultimate carcinogen of benzo[a]pyrene. Even though the mechanism of the differences in the activities of hGSTP1-1 variants toward anti-5-MeCDE versus anti-BPDE remains to be elucidated, it seems that the molecular configuration of the diol epoxide is an important determinant of the activity of hGSTP1-1 isoforms toward polycyclic aromatic hydrocarbon diol epoxides.

Characterization of DNA adducts formed by the four configurationally isomeric 5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxides.

The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.

Effect of 2-chloro-4-methylthiobutanoic acid in a rapid bioassay for gastric carcinogens.

2-Chloro-4-methylthiobutanoic acid (CMBA, a mutagen from Japanese salted fish) at 15-500 mg/kg body weight induced several-fold increase in replicative DNA synthesis (RDS) (P < 0.05) after 80 min and 17 h, equivocal unscheduled DNA synthesis (UDS) after 80 min and necrosis 80 min after its administration in the stomach pyloric mucosa of F344 and ACI male rats. A positive control, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 50 mg/kg body weight), induced RDS, UDS and erosion. However, the negative control L-methionine (500 mg/kg body weight) did not display any effect. The results suggest possible tumor-initiating and -promoting activity of CMBA but at a lower potency than that of MNNG.

Mammary carcinogenicity of diol epoxide metabolites of benzo[j]fluoranthene in female CD rats.

The mammary carcinogenicity of two diol epoxide metabolites of the commonly occurring environmental carcinogen benzo[j]fluoranthene (BjF) was investigated by direct application to the tissue beneath the mammary glands of female CD rats. The compounds tested were trans-4,5-dihydroxy-anti-6,6a.epoxy-4,5,6,6a-tetrahydroBjF (BjF-4,5-DE) and trans-9,10-dihydroxy-anti-11, 12-epoxy-9,10,11,12-tetrahydroBjF (BjF-9,10-DE). The positive control was trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthren e (BcPDE). Groups of 20 female CD rats were maintained on AIN-76A-based high fat diet (23.5% corn oil) and at age 30 days were given three injections of 0.2 mumol of each compound in 0.1 ml dimethyl sulfoxide (DMSO), or DMSO alone, in the tissue underlying each of the three left thoracic nipples. The three right nipple areas were injected with DMSO alone. On the next day, each rat received three injections of 0.2 mumol of each compound in 0.1 ml DMSO, or DMSO alone, under each of the three left inguinal nipples, and DMSO alone under the three right nipples. The experiment was terminated after 44 weeks. BjF-9,10-DE, mean latent period 21.0 +/- 8.8 weeks, was more active than BjF-4,5-DE, mean latent period 36.2 +/- 8.0 weeks. BjF-9,10-DE induced tumors in 70% of the rats; a total of 38 fibroadenomas and eight adenocarcinomas was observed. BjF-4,5-DE induced tumors in 55% of the rats. These included 17 fibroadenomas, seven dysplastic fibroadenomas, and two adenocarcinomas. BcPDE induced tumors rapidly, with a mean latent period of 9.7 +/- 4.0 weeks. All BcPDE-treated rats had mammary tumors. A total of 46 adenocarcinomas, as well as other tumors, were observed. In the DMSO-treated rats, mammary tumor incidence was 15%. The results of this study demonstrate that BjF-9,10-DE is more carcinogenic in the rat mammary gland than BjF-4,5-DE and that low doses of both diol epoxide metabolites of BjF are effective mammary tumorigens in female CD rats maintained on a high fat diet.

Mammary carcinogenicity in female CD rats of fjord region diol epoxides of benzo[c]phenanthrene, benzo[g]chrysene and dibenzo[a,l]pyrene.

We compared the mammary carcinogenicity in female CD rats of three fjord region diol epoxides to test our hypothesis that such sterically hindered molecules would be potent carcinogens. The diol epoxides tested were racemic anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPDE), anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo[g]chrysene (BgCDE) and anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l ]pyrene (DB[a,l]PDE). Each diol epoxide was dissolved in dimethylsulfoxide (DMSO) and injected under the six nipples on the left side of the rat, with DMSO only being injected under the nipples on the right side. The total dose of each diol epoxide was 1.2 mumol/rat and there were 20 rats/group. The experiment was terminated 41 weeks after treatment. All three diol epoxides were potent mammary carcinogens, with activity greater than previously observed for a bay region diol epoxide, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). DB[a,l]PDE induced tumors most rapidly, followed by BcPDE and BgCDE. However, different types of tumors were induced. For induction of adenomas and adenocarcinomas, BcPDE and BgCDE had comparable potency; both were more active than DB[a,l]PDE. In contrast, for induction of sarcomas, DB[a,l]PDE was significantly more active than BcPDE and BgCDE. The results of this study support our hypothesis that sterically hindered fjord region diol epoxides are potent mammary carcinogens in the rat.

Convenient synthesis of 3-methoxybenz[a]anthracene-7,12-dione.

The regioselective synthesis of 3-methoxybenz[a]anthracene-7,12-dione by oxidative photocyclization is described. The synthesis involved preparation of 1-(1-bromo-2-naphthyl)-2-(3- methoxyphenyl)ethylene by Wittig reaction, followed by photocyclization for 4 h. This gave 7-bromo-3-methoxybenz[a]anthracene. Extended photocyclization over 16 h under similar conditions gave 3-methoxybenz[a]anthracene-7,12-dione, an important intermediate in the synthesis of 7,12-dimethylbenz[a]anthracene-3,4-diol 1,2-epoxide.

Synthesis of Fjord region diol epoxides as potential ultimate carcinogens of dibenzo[a,l]pyrene.

Dibenzo[a,l]pyrene (DB[a,l]P) is one of the strongest polynuclear aromatic hydrocarbon carcinogens known. This paper describes the synthesis of potential ultimate carcinogens of DB[a,l]P: anti- and syn-11,12-dihydroxy-13,14-epoxy-11,12,13,14- tetrahydroDB[a,l]P (DB-[a,l]P-11,12-diol-13,14-epoxides). The method employed is also useful for the preparation of key intermediates for the synthesis of 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo-[g]chrysene (BgC-11,12-diol-13,14-epoxide). Photochemical cyclization of the appropriately substituted phenanthrylphenylethylenes provided 9-carbomethoxy-11-methoxyBgC (4) and 11-methoxyBgC (3). The former was converted by reduction, oxidation, one-carbon chain extension, and cyclization to 11-methoxy DB[a,l]P (7). Compounds 3 and 7 were converted by hydrolysis and oxidation to BgC-11,12-dione (10) and DB[a,l]P-11,12-dione (11), respectively. The diones are the precursors for the synthesis of the corresponding diol epoxides. anti- and syn-DB-[a,l]P-11,12-diol-13,14-epoxides 13 and 14 were prepared in 38% and 55% yields, respectively, from 11. Both diol epoxides had predominantly pseudodiequatorial hydroxyl groups, as seen in other sterically hindered diol epoxides.

Formation of the acrolein-derived 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with 3-(N-carbethoxy-N-nitrosamino)propionaldehyde.

3-(Methylnitrosamino)propionaldehyde (MNPA) is a carcinogenic nitrosamine formed by nitrosation of arecoline, a major alkaloid in areca nut which is a constituent of betel quid. While DNA adducts of its analogue, 3-(methylnitrosamino)propionitrile, have been characterized, little is known about the structures of DNA adducts by MNPA. In this paper, we report that the acrolein-derived 1,N2-propanoguanine adducts are formed upon incubating deoxyguanosine or DNA with 3-(N-carbethoxy-N-nitrosamino)propionaldehyde, a stable carbamate precursor of the metabolically activated MNPA. The identities of these adducts were confirmed by HPLC co-migration, by their NMR and UV spectra, and by chemical properties as compared with those of the synthetic standards. Analogous results were obtained from the reaction of the carbamate with calf thymus DNA. Upon acid or enzyme hydrolysis of the carbamate-modified DNA, acrolein-guanine adducts were detected, and the levels were quantitated. Again, the identities of the adducts were verified by co-chromatography with the standards, by UV spectroscopy, or by the ring-opening with NaOH/NaBH4. Consistent with its ability to modify DNA, the carbamate was found to be mutagenic in Salmonella tester strains. These results show that acrolein is a likely metabolite from the activation of MNPA and that the formation of 1,N2-propanoguanine adducts may contribute to the mutagenicity of the carbamate of MNPA.

Metabolic effect of orotic acid in calves.

Eight calves (males, Black and White crossbred with Holstein-Fresian) were fed milk and milk replacer without (control group) or with potassium orotate (3 mmol./l.) supplementation for 6 weeks after birth. Orotate depressed the biosynthesis of polyamines in mucosa of the gastrointestinal tract (rumen, omasum, abomasum, colon) by decreasing of ornithine decarboxylase activity with a simultaneous compensatory increase of S-adenosyl-methionine decarboxylase activity. A lower concentration of spermidine and spermine in the mucosa of the colon was also noted. The above changes were accompanied by increased urinary excretion of ornithine and arginine. Calf adaptation to a high OA intake was associated with an increased activity of the OA metabolizing enzyme complex (orotate phosphoribosyl transferase and orotidine monophosphate decarboxylase) in the liver, while urinary OA losses diminished with age. Increased concentrations of uracil and uridine in the liver and higher urinary excretion of pseudouridine in OA-fed calves was also observed. Stimulation of pyrimidine metabolism by OA depressed purine synthesis, which was reflected by a decrease of urate, hypoxanthine, and xanthine concentration in the liver. Interestingly OA enhanced urate excretion by the kidneys. OA strongly affected lipid metabolism in calves because total cholesterol, LDL-, and HDL-cholesterol, and triglycerides in blood plasma decreased while triglycerides accumulated in the liver of OA-fed calves. Milk OA in concentrations characteristic of cows with hereditary orotic aciduria exerts an unfavourable effect on the metabolism of polyamines, purines, and lipids in calf tissues.

Variability of orotic acid concentration in cow's milk.

The average concentration of orotic acid in the milk of 412 Black and White bred cows from four Polish provinces was 0.618 +/- 0.233 mmol/l. There was no correlation between milk yield and concentration of orotic acid. A higher concentration of this pyrimidine in younger cows, and its increase during development of lactation was noted. The yearly pattern of orotic acid in milk and urine of four low-, and four high-orotate cows was examined. In spite of high average differences in milk orotate (0.397 and 0.813 mmol/l) no significant differences in urinary orotate (20.96 and 21.90 mumol/mmol creatinine) were observed. In both groups the lowest milk orotate level occurred in early lactation. The orotic acid content (mmol/l) in commercial milk products was as follows: skim milk--0.783; evaporated milk--0.538; cream 12% fat--0.367; buttermilk--0.449; yogurt--0.331; kefir--0.341; sour milk 2% fat--0.360; dried skim milk--1.042; Bebiko I (infant formula)--0.650. Leukemia led to the elevation (0.845 mmol/l), whereas mastitis to the depression (0.124 mmol/l) of milk orotic acid level.

Intracranial pressure monitoring in the posterior fossa: a preliminary report.

Direct therapeutic drainage and intracranial pressure monitoring from the posterior fossa has never been accepted in neurosurgical practice. Potential complications including cerebrospinal fluid leak, cranial nerve palsies, and brain-stem irritation have been a major deterrent. The authors placed a catheter for pressure monitoring in the posterior fossa of 20 patients in the course of posterior fossa surgery: 14 patients with acoustic schwannomas, four with posterior fossa meningiomas, one with cerebellar hemangioblastoma, and one with a solitary cerebellar metastatic lesion. A Richmond bolt was also placed in the frontal area. Continuous monitoring of the supratentorial and infratentorial compartments was performed for 48 hours. During the first 12 hours the posterior fossa pressure was 50% greater than that of the supratentorial space in all patients (p less than 0.01). Over the next 12 hours the supratentorial pressure was 10% and 15% higher than the posterior fossa pressures in all patients, and by 48 hours of monitoring the pressures had equilibrated. There was no mortality or morbidity referable to insertion of the posterior fossa catheter. The conclusions drawn from this study are that: 1) direct monitoring and drainage of the posterior fossa is safe and effective; and 2) within the early postoperative period, the supratentorial pressures failed to reflect what is taking place within the posterior fossa. The implications and advantages of direct posterior fossa monitoring in the postoperative patient are discussed.

Temporary motor and sensory paralysis associated with intrathecal administration of morphine.

We are reporting a temporary, totally reversed motor and sensory paralysis subsequent to the intrathecal administration of 1.6 mg of morphine sulfate. This may represent an event which is not based on medication-induced myelopathy but on cardiovascular changes occurring as a result of pain relief.

Hospitalization on the high-risk maternity unit. A pilot study.

Women hospitalized for high-risk pregnancy appear to be at high risk for adverse reactions to hospitalization. Nine pregnant women remaining in hospital for 1 week or more were studied prospectively with a semistructured questionnaire, standard self-administered measures, and staff rating. Six women presented serious problems of compliance or psychic functioning. Women who suffered these adverse reactions tended to be poor, unemployed, younger, less educated, Catholic, unmarried, and to have children at home, when compared to the three women not experiencing difficulty. Women subsequently manifesting problems were significantly likelier on admission to appear as "cases" on the SCL-90-R and to have higher scores on the POMS. On the basis of pilot results, hospitalization for high-risk pregnancy appears to be a particularly stressful event. Women most at risk for problems can probably be identified prospectively.

Protein supplement resistant against ruminal degradation as a factor improving utilization of urea in ruminant feeding.

The metabolic and productive effects of the blood meal and formaldehyde (FA) treated casein supplements (5-10% of crude protein content) given with urea concentrates in sheep and fattening bulls were investigated. The blood meal has a similar composition of essential amino acids (EAA) to casein. The mean solubility of the FA treated casein and the blood meal after 6 hours of incubation in the sterilized rumen contents amounted 10.5% and 8.5% respectively. The average rumen ammonia concentration and plasma urea level was the highest in bulls fed urea ration without protected protein supplement. The supplementation of this ration with blood meal diminished the large daily fluctuation of plasma AA level and increased plasma EAA/NEAA ratio. The blood meal supplement improved the nitrogen retention in sheep (14%) and body gains in bulls (9%) but did not influence digestible coefficients and rumen protein synthesis in sheep.