Phenoxyethanol: protein preservative for taxonomists.

Pieces of chicken heart or skeletal muscle were placed in a dilute solution of the antimicrobial agent 2-phenoxyethanol and stored at room temperature. Under these conditions, the serum albumin, lactate dehydrogenase, and malate dehydrogenase in these tissues survived in easily detectable amounts for at least 2 weeks. The surviving proteins appeared to be identical with those of fresh tissues in physical, catalytic, and immunological properties. Phenoxyethanol also preserved heart and muscle proteins of representatives of other vertebrate classes. Tissue samples collected in the analysis by biochemical taxonomists.

Abnormal regulation of renal kallikrein in experimental diabetes. Effects of insulin on prokallikrein synthesis and activation.

The effects of streptozotocin (STZ) diabetes and insulin on regulation of renal kallikrein were studied in the rat. 1 and 2 wk after STZ injection, diabetic rats had reduced renal levels and urinary excretion of active kallikrein. Tissue and urinary prokallikrein levels were unchanged, but the rate of renal prokallikrein synthesis relative to total protein synthesis was reduced 30-45% in diabetic rats. Treatment of diabetic rats with insulin prevented or reversed the fall in tissue level and excretion rate of active kallikrein and normalized prokallikrein synthesis rate. To further examine insulin's effects, nondiabetic rats were treated with escalating insulin doses to produce hyperinsulinemia. In these rats, renal active kallikrein increased. Although renal prokallikrein was not increased significantly by hyperinsulinemia, its synthesis was increased. As this was accompanied by proportionally increased total protein synthesis, relative kallikrein synthesis rate was not changed. Excretion of active kallikrein was unchanged, but prokallikrein excretion was markedly reduced. Therefore, increased tissue active kallikrein seen with hyperinsulinemia can be explained not only by increased synthesis but also by retention and increased activation of renal prokallikrein. These studies show that STZ diabetes produces an impairment in renal kallikrein synthesis and suggest that this disease state also impairs renal prokallikrein activation. The findings also suggest that insulin modulates renal kallikrein production, activation, and excretion.

Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol.

Indole-3-carbinol (I3C), a natural constituent of cruciferous vegetables, is an inhibitor in several experimental animal models of carcinogenesis by polynuclear aromatic hydrocarbons or aflatoxin B1 (AFB1) when administered prior to or during carcinogen exposure. For assessment of the postinitiation effects of I3C, rainbow trout were exposed to dietary I3C in two different protocols--before and during AFB1 exposure or after AFB1 exposure only. Preinitiation exposure to I3C reduced AFB1-initiated hepatocellular carcinomas in trout as previously reported, but post-initiation I3C exposure strongly enhanced the tumor incidence above the positive AFB1 control. These results reveal the need for additional research to elucidate the overall effect of I3C on chemical carcinogenesis.

Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection.

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

Promotion of aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency.

Indole-3-carbinol (13C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout (Bailey et al., J. Natl. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick et al., unpublished results) when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human 13C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed 13C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, 13C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of 13C treatment. Promotional enhancement of tumor incidence by 13C was found to be significant when 13C treatment was delayed for several weeks or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to 13C treatment and to be decreased but still evident when 13C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify 13C promotional potency in terms of its dietary concentration, a series of AFB1 tumor dose-response curves was established, each with a different level of 13C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing 13C concentration. The level of 13C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm 13C, fed continuously, with no substantial threshold for promotion. By comparison, 13, when fed before and with AFB1, shows a 50% inhibitory value (13C concentration that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm 13C [Dashwood et al., Carcinogenesis (Lond.), 10: 175-181, 1989]. Thus the potential for 13C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.

Anticarcinogenic activity of indole-3-carbinol acid products: ultrasensitive bioassay by trout embryo microinjection.

The relative contribution of indole-3-carbinol (I3C) and its acid condensation products to the anticarcinogenic activity of this crucifer phytochemical has been studied using trout embryo microinjection. I3C was treated with 0.07 N HCl to give a reaction mixture (RXM) comprising < 0.5% parent compound and over 20 products, the most prevalent being the dimer 3,3'-diindolylmethane (I33') and a related cyclic trimer (CT). RXM, I33' or CT was injected into embryos with [3H]aflatoxin B1 (AFB1) and total embryonic DNA was isolated 1, 3, or 10 days postinjection. Compared with controls given AFB1 alone, I3C failed to inhibit carcinogen-DNA binding at any time point. In contrast I33', CT, and RXM inhibited AFB1-DNA binding by an average of 37, 51, and 65%, respectively. Coinjection of AFB1 and 350 microM I3C, RXM, or I33' into trout embryos reduced AFB1-induced hepatocarcinogenesis after 1 year from 43.4% in positive controls to 36.0, 12.2 (P < 0.05), and 24.6% (P < 0.05), respectively. No tumor data were obtained in the AFB1 plus CT group due to poor survival of the embryos posthatching. These results indicate that acid condensation products, not the parent compound, represent the anticarcinogenic species in trout and that their formation in the stomach is a likely prerequisite for I3C anticarcinogenesis.

The purification of a bovine kidney enzyme which cleaves melanocyte-stimulating hormone-release inhibiting factor.

An enzyme which catalyzes the hydrolysis of L-prolyl-L-leucylglycinamide, the factor which inhibits the release of melanocyte-stimulating hormone, was purified 189-fold from bovine kidney in a 5% yield. The molecular weight of the enzyme on gel filtration was estimated to be 300 000 and its isoelectric point was found to be pH 4.1. The single component seen on sodium dodecyl sulphate-gel electrophoresis was estimated to have a molecular weight of 56 000, indicating that the native enzyme may be a pentamer or hexamer. The enzyme could clearly be distinguished from other prolyl-cleaving enzymes.

Purification and specificity of prolyl dipeptidase from bovine kidney.

Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.

An improved method of isolation of rat pancreatic prokallikrein. Characterization of the zymogen and of the kallikrein produced by trypsin activation.

A prokallikrein was purified 1600-fold from rat pancreatic tissue in an overall yield of 40% by a simple four-stage procedure. The final and crucial step was immunoaffinity chromatography utilizing antibody raised to a very small amount of prokallikrein. Both the pure zymogen and the active kallikrein generated from it by trypsin activation are single chain species with Mr values of 38400 +/- 300 and 35500 +/- 400, respectively. Valine is the N-terminal amino acid residue of prokallikrein. The zymogen was comparatively stable both to autoactivation and denaturation with respect to temperature and pH. The kallikrein produced by trypsin activation of the zymogen was similar in some of its catalytic properties to the kallikrein purified from autolyzed rat pancreas but the two species differed in their susceptibility to substrate activation.

Purification of prokallikrein from bovine pancreas.

A prokallikrein was isolated from bovine pancreas by a multi-step procedure involving gel filtration, hydrophobic interaction and anion-exchange chromatographies. The purification was initially monitored by measurement of the kinin-releasing activity of the activated zymogen. Later, when the pure prokallikrein had been isolated, a specific radioimmunoassay for the zymogen was set up and that was employed to provide estimates of 323-fold and 28% for the overall degree of purification and percentage recovery of prokallikrein. The relative molecular weight of prokallikrein was found to be 26,900 by SDS gel electrophoresis and its isoelectric point was established as pH 4.55.

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

Purification and characterization of a fibrinogenase from the venom of Western Diamondback rattlesnake (Crotalus atrox).

A fibrinogenolytic enzyme was isolated from the venom of Western Diamondback rattlesnake (Crotalus atrox) by a three-step procedure involving gel filtration and anion-exchange chromatography. The molecular weight was estimated as 22 900 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 4.65. The enzyme rapidly destroyed the ability of bovine fibrinogen to form a clot on incubation with thrombin. Incubation of fibrinogen with the fibrinogenolytic enzyme for 5 min resulted in the disappearance of the beta-chain of fibrinogen and the appearance of lower molecular weight fragments. Thus the enzyme can be classified as a beta-fibrinogenase. However, on prolonged incubation of the fibrinogen there was also a partial digestion of the alpha-chain. The fibrinogenase showed no activity towards fibrin or casein or arginine esters. The fibrinogenolytic activity was inhibited by phenylmethanesulphonyl fluoride (PMSF) but was unaffected by EDTA.

A 1H-NMR study of the interactions between rat tissue kallikrein and two peptide inhibitors.

The 1H-NMR spectra have been obtained for rat submandibular kallikrein in the absence and presence of inhibitors. Two competitive inhibitors were investigated, the tripeptide leupeptin (a potent inhibitor with Ki 0.5 microM) and a hexapeptide (a much weaker, substrate-analogue inhibitor with Ki 380 microM). Analysis of the NMR spectra showed that binding of leupeptin to kallikrein led to a change in the conformation of the enzyme, whereas binding of the substrate analogue to the enzyme produced no such change and may have resulted in a conformational change of the inhibitor.

Immunological properties of nerve growth factors.

Antisera were raised against nerve growth factors isolated from mouse salivary gland and five venoms representative of the three main families of poisonous snakes. Immunochemical cross-reactivity was assessed from the ability of the antisera to inhibit the biological activities of the heterologous antigens. The high and low molecular weight forms of the salivary gland factors were found to be immunologically identical but distinct from the snake venom factors. The snake venom factors, on the other hand, were immunologically closely related to each other but not identical.

Urinary and renal tissue kallikrein in the streptozocin-diabetic rat.

The renal kallikrein-kinin system is thought to participate in blood pressure regulation and displays abnormalities in human hypertension, as well as in many animal models of hypertension. Urinary excretion and tissue levels of renal kallikrein were measured in streptozocin (STZ)-diabetic rats in relation to blood pressure, glycemia, and insulin treatment. In study 1, STZ-diabetic rats with marked hyperglycemia showed reduced kallikrein-like esterase excretion, compared with control rats, when first measured after 7 days of diabetes (9.9 +/- 2.5 versus 17.5 +/- 2.4 EU/24 h, P less than 0.05). This difference increased with time and, after 210 days, urinary esterase excretion in diabetic and control rats was 6.7 +/- 2.1 and 39.0 +/- 6.0 EU/24 h, respectively (P less than 0.001). Urine kallikrein, measured by radioimmunoassay, was similarly reduced in diabetic rats (40.4 +/- 8.0 versus 88.0 +/- 6.5 micrograms/24 h, at 30 days, P less than 0.001). At 120 days, systolic blood pressures were elevated in diabetic rats (P less than 0.05), and at 180 days over 60% of the diabetic rats had pressures above the highest pressures of control rats. In study 2, STZ-diabetic rats were treated with insulin for 2 wk (2 U NPH at 0800 h, or 2 U NPH at 0800 and 1600 h). In the single-dose group, with hyperglycemia similar to that of diabetic rats in study 1, kallikrein excretion was reduced as early as day 2, compared with nondiabetic rats (56.0 +/- 6.1 versus 109 +/- 9.4 micrograms/24 h, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term, high-dose dietary exposure of rainbow trout to butylated hydroxyanisole is non-carcinogenic.

The hepatocarcinogenic and/or promotional properties of butylated hydroxyanisole (BHA) were tested in rainbow trout. Four groups of 100, 21-day-old trout embryos were exposed to 0.5 ppm aqueous aflatoxin B1 (AFB1) for 30 min, and four similar groups were sham treated. After hatching, swim-up, and the onset of vigorous feeding behavior (2 weeks pest swim-up), duplicate groups of 60 AFB1-treated and sham-treated fry were started on a test diet containing 0.6% (6000 ppm) BHA, and the other duplicate groups of treated and untreated fry were fed the control Oregon Test Diet (OTD). After 8 months of feeding BHA or OTD, the fish were necropsied for tumor detection, with particular attention given to the stomach and liver. No tumors were seen in the livers or stomachs of the sham-treated fish fed OTD or BHA, showing that BHA is not carcinogenic to rainbow trout under the conditions of this experiment. Promotional results were equivocal, with one tank of fish having a higher hepatic tumor incidence, but the other the same as the positive AFB1 control. When the tanks were combined, however, there was no statistical difference between the two groups.

Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity.

We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000-65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10-12 micrograms kinin/hr/mg but released only 2 micrograms kinin/hr/mg at pH 7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Fish models for environmental carcinogenesis: the rainbow trout.

Progress over the past 30 years has revealed many strengths of the rainbow trout as an alternative model for environmental carcinogenesis research. These include low rearing costs, an early life-stage ultrasensitive bioassay, sensitivity to many classes of carcinogen, a well-described tumor pathology, responsiveness to tumor promoters and inhibitors, and a mechanistically informative nonmammalian comparative status. Low-cost husbandry, for example, has permitted statistically challenging tumor study designs with up to 10,000 trout to investigate the quantitative interrelationships among carcinogen dose, anticarcinogen dose, DNA adduct formation, and final tumor outcome. The basic elements of the trout carcinogen bioassay include multiple exposure routes, carcinogen response, husbandry requirements, and pathology. The principal known neoplasms occur in liver (mixed hepatocellular/cholangiocellular adenoma and carcinoma, hepatocellular carcinoma), kidney (nephroblastoma), swim bladder (adenopapilloma), and stomach (adenopapilloma). Trout possess a complex but incompletely characterized array of cytochromes P450, transferases, and other enzymic systems for phase I and phase II procarcinogen metabolism. In general, trout exhibit only limited capacity for DNA repair, especially for removal of bulky DNA adducts. This factor, together with a high capacity for P450 bioactivation and negligible glutathione transferase-mediated detoxication of the epoxide, accounts for the exceptional sensitivity of trout to aflatoxin B1 carcinogenesis. At the gene level, all trout tumors except nephroblastoma exhibit variable and often high incidences of oncogenic Ki-ras gene mutations. Mutations in the trout p53 tumor suppressor gene have yet to be described. There are many aspects of the trout model, especially the lack of complete organ homology, that limit its application as a surrogate for human cancer research. Within these limitations, however, it is apparent that trout and other fish models can serve as highly useful adjuncts to conventional rodent models in the study of environmental carcinogenesis and its modulation. For some problems, fish models can provide wholly unique approaches.

Purification by HPLC anion-exchange chromatography and some properties of a kinin-releasing enzyme from the venom of Agkistrodon halys ussuriensis.

A kinin-releasing enzyme was isolated from the venom of Agkistrodon halys ussuriensis using a very convenient two-step procedure consisting of HPLC anion-exchange chromatography. The relative molecular mass was estimated as 24,000 by SDS gel electrophoresis and an isoelectric point of pH 3.7 was established by gel isoelectric focusing. The kinin-releasing enzyme resembled a typical tissue kallikrein in the values of such physiochemical parameters, in its thermal stability, in its lack of activity towards fibrinogen and casein, and in its behaviour with potential inhibitors. Thus, the enzyme can be classified as a kallikrein-like enzyme.