Histologic and Immunohistochemical Characterization of Pheochromocytomas in 20 Clouded Leopards ( Neofelis nebulosa).

Clouded leopards in North American zoological institutions have a high frequency of pheochromocytomas and were identified in 32 of 70 (45%) animals necropsied. Archival sections of adrenal gland from 20 adult clouded leopards with unilateral or bilateral pheochromocytomas collected between 1984 and 2011 were examined by light microscopy and immunohistochemistry, and case demographics were reviewed. Affected leopards were older than 10 years of age (mean, 16 years; range, 11-19 years), and males were overrepresented (12 males, 8 females). Pedigree analysis yielded no evidence for heritability. Five clouded leopards had bilateral neoplasms. Pheochromocytoma was the cause of death due to invasion of the caudal vena cava and fatal hemorrhage in 4 cases. Most pheochromocytomas were well-demarcated, nodular, and expansile masses composed of cords and packets of neoplastic polygonal cells. Five pheochromocytomas had vascular invasion, of which 4 resulted in hemorrhage that was the cause of death. One of the latter pheochromocytomas also had pulmonary metastasis. Ultrastructurally, neoplastic cells had cytoplasmic structures consistent with both norepinephrine- and epinephrine-containing granules. In all cases, neoplasms were immunohistochemically positive for chromogranin A, protein gene product 9.5, and synaptophysin. A subset of neoplasms evaluated by tissue microarray were positive for met-enkephalin and ß-endorphin and negative for melan-A. Histologically, 7 of 20 (35%) clouded leopards with pheochromocytomas had retinal detachment, retinal degeneration, or intramyocardial muscular arteriosclerosis, suggestive of hypertension. Pheochromocytomas can cause mortality and may be a source of clinically significant hypertension in clouded leopards. These neoplasms share similar histologic, immunohistochemical, and ultrastructural characteristics with those of other species.

The effects of Strongylus vulgaris parasitism on eosinophil distribution and accumulation in equine large intestinal mucosa.

REASONS FOR PERFORMING STUDY: Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. OBJECTIVE: To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. METHODS: Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. RESULTS: There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. CONCLUSIONS: Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.

The cloning and expression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase 2 in normal canine lymph nodes and in canine lymphoma.

Matrix metalloproteinase-2 (MMP-2) and its inhibitor, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), are known to be important in cancer. The purposes of this study were to determine the cDNA sequence of canine MMP-2 and to investigate the expression patterns of MMP-2 and TIMP2 in normal canine lymph nodes and spontaneously arising canine lymphomas. We cloned and sequenced a PCR product containing most (1901 base pairs) of the coding sequence of canine MMP-2 that translates into a 623 amino acid protein. The cDNA and deduced amino acid sequences are highly homologous to those of other mammalian species. Canine MMP-2 and TIMP2 mRNAs were detectable in the majority of normal lymph node and lymphomatous samples evaluated. No statistical difference was identified when comparing the expression of either gene with regard to normal versus neoplastic nodes, nodal versus extranodal lymphoma, lymphoma grade, or B versus T cell immunophenotype.

Evaluation of the safety and bioactivity of purified and semi-purified glucoraphanin.

The anti-carcinogenic effects of broccoli have been attributed to sulforaphane, the hydrolysis product of glucoraphanin (GRP). Here we determined if purified GRP, in the absence of the plant-derived hydrolyzing enzyme myrosinase, could affect pulmonary and hepatic ethoxyresorufin O-deethylase (EROD) and/or NAD(P)H-quinone oxidoreductase 1 (NQO1) activity. Male F344 rats were administered semi-synthetic, semi-purified or purified GRP (240 mg/kg: 550 micromol/kg rat daily for 4 days) by gavage. Hepatic and pulmonary NQO1 activity increased ( approximately 20%), but not EROD. Varying doses of semi-purified GRP (30, 60, or 120 mg/kg rat daily for 4 days) again caused no change in EROD activity, although a dose-dependent increase in NQO1 was seen. Urinary excretion of mercapturic acids showed no difference between preparations, and recovery increased with decreasing dose. Histopathologic examination revealed no abnormal tissues other than cecum, where inflammation was dose dependent; mild at 120 mg/kg and severe at 240 mg/kg, a greatly supra-physiological dose. We conclude that GRP 30-60 mg/kg p.o. is safe and effectively enhances NQO1 in all tissues evaluated.

Inhibition of liver carcinogenesis in Wistar rats by consumption of an aqueous extract from leaves of Ardisia compressa.

This study evaluates the chemopreventive effect of an aqueous extract of dried leaves of Ardisia compressa against liver cancer. A rat liver assay that mimics progressive forms of human disease was used as a carcinogenesis model. Forty-five male Wistar rats (180-200 g body weight) were injected intraperitoneally on day 1 with a single dose (100 mg/kg) of diethylnitrosamine (DEN), and also received via gavage 20 mg/kg acetylaminofluorene (2-AAF), on days 7, 8 and 9. The rats were randomly divided into four groups (n=15). Control groups (Group 1 and Group 2) had free access to water. Group 3 received 0.5% (w/v) of A. compressa tea for 10 days before treatment and during the study as the sole source of fluid until the rats were killed. A fourth group of 15 rats received no carcinogen or promoter but did receive 0.5%, (w/v) of A. compressa tea. All animals had 70% partial hepatectomy at day 10. The incidences of hepatocellular foci, nodules and carcinoma were significantly smaller in Group 3 than in Group 2 (P<0.01). A. compressa tea consumption alone (Group 4) did not induce the development of foci, nodules or carcinomas (P<0.01). The striking observation of this study was that consumption of A. compressa tea resulted in complete inhibition of the chemically-induced hepatocarcinogenesis in Wistar rats.

Luminal nutrients exacerbate intestinal hypoxia in the hypoperfused jejunum.

BACKGROUND: Provision of enteral nutrients shortly after traumatic injury has become the preferred method of nutrition support provided to patients. However, traumatic shock results in splanchnic hypoperfusion, which may cause persistent intestinal hypoxia. This study tested the hypothesis that delivery of enteral nutrients to the hypoperfused jejunum increases oxidative demand beyond that available, thereby exacerbating intestinal hypoxia. METHODS: Wistar-Furth rats (186+/-4 g; n = 24) were randomized to receive intestinal hypoxia (superior mesenteric artery occlusion) or serve as normoxic controls (sham laparotomy). Within the jejunum of each rat, 4 6-cm loops were randomized to receive luminal perfusions with 1 of 4 substrates: mannitol (an osmotic control); glucose (undergoes active transport via the sodium-glucose co-transporter [SGLT-1] and is metabolized); 3-o-methylglucose (3-o-mg; uses SGLT-1 but is not metabolized); or fructose (does not use SGLT-1 but is metabolized). After in situ perfusions, jejunal tissue was removed for analysis of nutrient transport and barrier function in modified Ussing chambers. Tissue homogenate was used to determine concentration of ATP, lactate, pyruvate, and protein. Also, jejunal tissue was stained with hematoxylin and eosin for qualitative analysis of ischemia and necrosis. RESULTS: Transmural resistance was lower (p < .001) in the hypoxia groups, irrespective of substrate, indicating increased mucosal permeability. When compared with the normoxic controls, glucose transport was impaired (p < .001) in the hypoxic groups; however, glutamine transport was unaffected. The degree of intestinal hypoxia, assessed by jejunal lactate concentration, was higher (p < .001) in the glucose and fructose groups, than the control mannitol and 3-o-mg groups. CONCLUSIONS: The observation that 3-o-mg did not differ from the mannitol control indicates that SGLT-1 activation alone does not exacerbate hypoxia. Rather, these results indicate that provision of metabolizable nutrients to the hypoperfused intestine exacerbate hypoxia and potentially lead to intestinal ischemia. Although early enteral nutrition is an important intervention after trauma, care must be taken to ensure intestinal perfusion is adequate to allow for nutrient metabolism and prevent further compromise.

Rapid isolation and purification of 1-cyano-2-hydroxy-3-butene (crambene) from Crambe abyssinica seed meal using immiscible solvent extraction and high-performance liquid chromatography.

1-Cyano-2-hydroxy-3-butene (crambene) is a nitrile found in cruciferous vegetables that causes significant upregulation of quinone reductase and glutathione S-transferases in vivo and in vitro, making it a likely candidate as a cancer chemopreventive compound. To investigate further the putative anticarcinogenic mechanisms of crambene, a compound of the highest possible purity is vital. Therefore, a rapid and effective method of purification of crambene is necessary to continue studies of its beneficial health effects. A rapid method to isolate and purify natural crambene from either Crambe abyssinica (crambe) seed or commercially processed crambe seed meal was developed using immiscible solvent extraction followed by high-performance liquid chromatography. Use of this methodology eliminated the need for time-consuming and relatively inefficient column chromatography, improved extraction efficiency, and resulted in higher purity than previously used methodologies. Elimination of trace amounts of fatty acid residues, unachievable with previous methodologies, also was accomplished.

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney.

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.

Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea.

An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-microm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.

Variation of glucosinolates in vegetable crops of Brassica oleracea.

Glucosinolates were evaluated in 5 groups and 65 accessions of Brassica oleracea (50 broccoli, 4 Brussels sprouts, 6 cabbage, 3 cauliflower, and 2 kale) grown under uniform cultural conditions. Glucosinolates and their concentrations varied among the different groups and within each group. The predominant glucosinolates in broccoli were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), 3-butenyl glucosinolate (gluconapin), and 3-indolylmethyl glucosinoate (glucobrassicin). Glucoraphanin concentration in broccoli ranged from 0.8 micromol g(-1) DW in EV6-1 to 21.7 micromol g(-1) DW in Brigadier. Concentrations of the other glucosinolates in broccoli varied similarly over a wide range. In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were sinigrin (8.9, 7.8, 9.3, and 10.4 micromol g(-1) DW, respectively) and glucobrassicin (3.2, 0.9, 1.3, and 1.2 micromol g(-1) DW, respectively). Brussels sprouts also had significant amounts of gluconapin (6.9 micromol g(-1) DW). Wide variations in glucosinolate content among genotypes suggest differences in their health-promoting properties and the opportunity for enhancement of their levels through genetic manipulation.

Carotene, tocopherol, and ascorbate contents in subspecies of Brassica oleracea.

Cruciferous vegetables contain high levels of vitamins that can act as antioxidants, compounds that may protect against several degenerative diseases. The edible portions of 50 broccoli and 13 cabbage, kale, cauliflower, and Brussels sprouts accessions were assayed to determine variation in alpha-carotene, beta-carotene, alpha-tocopherol, gamma-tocopherol, and ascorbate contents within and between subspecies of Brassica oleracea. Ascorbate content was estimated in fresh samples using HPLC. Tissues for carotene and tocopherol analysis were lyophilized prior to extraction. Carotene and tocopherol concentrations were simultaneously measured using a reverse phase HPLC system. Results indicate that there is substantial variation both within and between subspecies. Kale had the highest levels of vitamins, followed by broccoli and Brussels sprouts with intermediate levels and then by cabbage and cauliflower, with comparatively low concentrations. Variability in vitamin content among the broccoli accessions suggests that potential health benefits that accrue with consumption are genotype dependent.

Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds.

The anti-tumor promoting activity of a polyphenolic fraction from grape seeds (GSP) was examined in CD-1 mouse skin epidermis. Specifically, the ability of this fraction to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and two markers of promotion in mouse skin, ornithine decarboxylase (ODC) and myeloperoxidase (MPO) activities, was evaluated. Pretreatment of mouse skin with 5, 10, 20 and 30 mg of GSP resulted in a dose-dependent reduction in TPA-induced epidermal ODC activity of 27, 37, 48 and 70%, respectively, compared to controls. In addition, pretreatment of mouse skin with 1, 5, 10 and 20 mg of GSP resulted in a significant 43, 39, 54 and 73% inhibition of MPO activity, respectively, compared to controls. In 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice, biweekly treatment of mouse skin with 5, 10, and 20 mg of GSP 20 min prior to TPA application resulted in a 30, 40, and 60% inhibition of final skin tumor incidence, respectively, compared to controls. In addition, the final number of tumors per mouse in the 5, 10 and 20 mg GSP-treated animals was decreased 63, 51, and 94%, respectively, compared to controls. These studies indicate that GSP possesses anti-tumor promoting activity when applied to CD-1 mouse skin prior to treatment with TPA. The mechanism of this tumor inhibition is due, in part, to a GSP-associated inhibition of TPA-induced epidermal ODC and MPO activities. Thus, GSP warrants further evaluation as a skin cancer chemopreventative agent.

Cutaneous antimicrobial preparation prior to intravenous catheterization in healthy dogs: clinical, microbiological, and histopathological evaluation.

The purpose of this study was to determine the effects of a one-minute chlorhexidine gluconate skin preparation protocol prior to cephalic vein catheterization. Twenty-three healthy beagle dogs had one leg aseptically prepared and the opposite leg served as a control. Twenty-six- and 77-hour time groups were studied. Chlorhexidine-treated legs had significantly lower cutaneous bacterial counts than the control legs prior to catheter insertion and prior to catheter withdrawal for both time groups. Control legs developed significantly more dermatitis than the treated legs after 77 h. A one-minute preparation with 4% chlorhexidine gluconate was an effective method for sustained reduction of cutaneous bacterial counts at peripheral intravenous catheter insertion points in dogs. Increased cutaneous bacterial counts were associated with significantly more microscopic dermatitis in untreated legs after 77 h of catheterization.

Xenobiotic metabolism, oxidant stress and chronic pancreatitis. Focus on glutathione.

Chronic pancreatitis, although relatively rare in the Western World, is common in certain tropical zones where staple crops such as cassava are rich in cyanogenic glycosides. This paper reviews the evidence for a cyanide connection, with reference to experimental studies using another plant nitrile, crambene; and then examines the hypothesis that chronic pancreatitis represents a manifestation of uncoordinated detoxification reactions between pancreatic cytochrome P450 mono-oxygenases and phase II conjugating enzymes, resulting in the irreversible consumption of glutathione in the acinar cell. The conclusion is that the central role of disrupted pancreatic glutathione status, as a result of 'xenobiotic stress', in the evolution of chronic pancreatitis cannot be overestimated. This position contrasts with that in acute pancreatitis, in which glutathione depletion has a pivotal role too, but occurs as a result of 'stress' from reactive oxygen species.

Characterization of rat pancreatic glutathione S-transferases by chromatofocusing, reverse-phase high-performance liquid chromatography, and immunohistochemistry.

The cytosolic glutathione S-transferases (GSTs) are a family of phase II detoxifying isoenzymes that catalyze the interaction of the tripeptide thiol glutathione (GSH) with a wide variety of reactive and often toxic or carcinogenic electrophilic substrates. Pancreatic GSTs, however, have only been partially characterized. In this study, pancreatic cytosolic GSTs from male Fisher 344 rats were semipurified by affinity chromatography and then analyzed for isoenzyme content by chromatofocusing (fast protein liquid chromatography) and for subunit content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. In addition, polyclonal rabbit antisera were produced against homodimeric isoenzymes purified from rat liver and kidney, including the alpha class isoenzymes 1-1 and 2-2, the mu class isoenzyme 4-4, and the pi class isoenzyme 7-7. These antisera were used in immunohistochemical (IHC) studies of the distribution of the pancreatic GSTs. A range of 0.5-1.6% of the total protein in rat pancreatic cytosol was found to be GST protein. The most abundant subunits present were the pi subunit 7 and mu subunits 3 and 4. Using modified methodology, smaller amounts of the alpha subunit 2 and the mu subunit 6 were detected, whereas very small amounts of the alpha subunits 1 and 8 were present. The IHC demonstrated that the GSTs were in large part limited to the duct system of the exocrine pancreas, with positive staining of endothelial cells and stroma observed for the alpha and mu subunits. Isoenzymes containing the alpha subunit 2 were preferentially expressed in centroacinar cells and small ductules, whereas those containing the mu subunit 4 and the pi subunit 7 were more prevalent within larger ductules and ducts. The lumens of the largest ducts also contained the two subunits 4 and 7. It is concluded that the acinar cells of the exocrine pancreas may lack the protection against electrophilic toxic and carcinogenic agents provided by the ductular system by GSTs.

Induction of rat pancreatic glutathione S-transferase and quinone reductase activities by a mixture of glucosinolate breakdown derivatives found in Brussels sprouts.

The chemoprotective effects of cruciferous vegetables against cancer has been linked to the induction of detoxification enzymes, including the phase II enzymes, glutathione S-transferases (GST) and quinone reductase (QR). Four glucosinolate breakdown products found in Brussels sprouts and previously shown individually to affect detoxification enzymes--(1-cyano-2-hydroxy-3-butene (Crambene), indole-3-carbinol (I3C), phenylethyl isothiocyanate (PEITC) and 1-isothiocyanato-3-(methylsulfinyl)-propane (IBN) were administered to male F344 rats by oesophageal intubation for 7 days both as a mixture and individually to assess the effect of these compounds on GST and QR activity in the pancreas, an organ previously shown to be affected by cruciferous diets. The doses of each compound in the mixture (50 mg Crambene/kg, 56 mg I3C/kg, 0.1 mg PEITC/kg and 38 mg IBN/kg) were chosen to represent the relative proportions of the parent glucosinolate for each compound in Brussels sprouts and shown to be below the toxic threshold for all the compounds. In rats receiving the mixture, pancreatic QR and GST activities were elevated 31- and 1.7-fold, respectively, while glutathione (GSH) was elevated threefold. On an individual basis, Crambene alone caused a 21-fold elevation of QR and 1.5-fold elevation of GST activities, while pancreatic GSH was elevated by both Crambene and PEITC 2.6- and twofold, respectively. No other significant effects of individual components were found. When the mixture was administered at 60% of the original dose, pancreatic QR and GST activities were elevated 12- and 1.4-fold, respectively, and pancreatic GSH was elevated 1.5-fold. At 20% of the original dose, pancreatic GSH was unaffected and QR and GST activities were elevated 2.7- and 1.3-fold, respectively. The results of these studies suggest that a diet rich in cruciferous vegetables may produce phase 11 enzyme induction in the pancreas, and that Crambene may be the most active component.

Effect of the beta-diketones diferuloylmethane (curcumin) and dibenzoylmethane on rat mammary DNA adducts and tumors induced by 7,12-dimethylbenz[a]anthracene.

Curcumin is a beta-diketone constituent of the spice turmeric that possesses anticarcinogenic properties in several animal models. The present studies were conducted in order to identify beta-diketones structurally-related to curcumin that would be effective dietary blocking agents toward the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Of the beta-diketone compounds initially screened for their capacity to induce quinone-reductase (QR) activity in wild-type Hepa1c1c7 cells and a mutant subclone, curcumin (diferuloylmethane) and dibenzoylmethane were most effective. However, when added to semipurified diets fed to female rats, dibenzoylmethane (1%), but not curcumin (1%), was effective in inhibiting in vivo mammary DMBA-DNA adduct formation. This inhibitory effect on mammary adduct formation was associated with a significant increase in liver activities of glutathione S-transferase, QR and 7-ethoxyresorufin-O-deethylase activities. Female rats provided diets supplemented with dibenzoylmethane at 0.1, 0.5 and 1.0% for 14 days prior to dosing with DMBA exhibited a significant decrease in mammary tumor development, compared with controls. However, tumor development for animals fed diets containing 1.0% curcumin was not different from that of controls. Therefore, dibenzoylmethane, and possibly other structurally-related beta-diketones, warrant examination as breast cancer chemopreventative blocking agents.

Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.

1-Cyano-2-hydroxy-3-butene (CHB) has been reported to cause cell death in rat pancreatic acini. In this report, we describe the time-dependent effects of CHB on mouse acinar cell apoptosis and the effects of CHB-induced acinar cell apoptosis on the severity of secretagogue-induced acute pancreatitis in mice. CHB administration to mice resulted in a time-dependent increase in pancreatic apoptosis, which was maximal 12 hours after CHB administration. The severity of pancreatitis was significantly reduced by prior CHB administration and maximal protection was observed when the caerulein injections were started 12 hours after CHB administration. These observations indicate that induction of apoptosis can reduce the severity of pancreatitis and they suggest that induction of pancreatic acinar cell apoptosis may be beneficial in the clinical management of acute pancreatitis.

The cruciferous nitrile, crambene, induces rat hepatic and pancreatic glutathione S-transferases.

Indoles and isothiocyanates found in cruciferous vegetables have been implicated as chemopreventive agents against carcinogenesis. The bioactivities of chemically related cruciferous nitriles, including 1-cyano-2-hydroxy-3-butene (crambene), however, have not been thoroughly evaluated. Crambene causes a prolonged elevation of rat hepatic and pancreatic glutathione and induces the GSH S-transferases (GSTs). Because elevated GST activity against the model substrate chlorodinitrobenzene does not reflect individual isoenzyme induction, quantitative HPLC evaluation of specific GST subunits is necessary to fully assess the range of GST isoenzymes induced by crambene. Accordingly, male Fischer 344 rats were given, via esophageal intubation, either 100 (Experiment 1) or 50 mg crambene/kg body wt (Experiment 2) once daily for 7 days. GSTs were extracted from hepatic cytosol by affinity chromatography, and the individual subunits that comprise the various isoenzymes were quantified by reverse-phase HPLC to gain an estimate of induction. In addition, pancreatic GST subunits were assessed in the low-dose experiment. In parallel with increased GST activity, crambene caused a generalized induction of GST subunits in both liver and pancreas, but the pattern of subunit induction was tissue dependent. In the liver, alpha subunits 1 and 2 and the mu subunit 3 were induced approximately 2-fold, while the mu subunit 4 was induced only 1.5-fold. In the pancreas, the alpha subunit 2 was induced to a much larger extent (2.6-fold) than the other subunits (from no induction to 1.6 fold). These results suggests that crambene-mediated GST induction mechanisms vary from tissue to tissue. Potential chemoprevention provided by crambene against GST-metabolized carcinogens or toxins may differ between liver and pancreas because of differences in the degree and pattern of induction.

A comparison of the individual and collective effects of four glucosinolate breakdown products from brussels sprouts on induction of detoxification enzymes.

Four glucosinolate derivatives were evaluated individually and as a mixture for their effects on hepatic P4501A (CYP1A), glutathione S-transferase (GST), quinone reductase (QR), glutathione reductase (G-Rd), and GSH levels. Doses of the derivatives were chosen to represent their relative abundance in Brussels sprouts. Adult male F344 rats received either corn oil (vehicle); one of the agents: indole-3-carbinol (I3C, 56 mg/kg), iberin (38 mg/kg), phenylethylisothiocyanate (PEITC, 0.1 mg/kg), or cyanohydroxybutene (crambene, 50 mg/kg); or all of the agents at the doses shown (as a mixture) given by gavage daily for 7 days. The mixture and I3C caused an 11- and 9.4-fold induction of CYP1A, respectively. Crambene and I3C each caused a 1.4-fold increase in GST, while the mixture caused a 2.5-fold increase. Crambene and I3C caused a 2.5- and 1.9-fold increase in QR, respectively. The mixture caused a 6.2-fold increase. Crambene, PEITC, and the mixture caused a 1.8-, 1.6-, and 2.0-fold increase in hepatic GSH levels, respectively. Crambene, I3C, iberin, and the mixture caused 1.3-, 1.4-, 1.2-, and 1.7-fold increases in G-Rd, respectively. In a second study the mixture was given at 60 and 20% of the original dose. CYP 1A, QR, G-Rd, and GST elevations were dose-dependent; GSH levels were not elevated. It is concluded that I3C and crambene are responsible for the majority of enzyme increases seen. A synergistic effect of I3C and crambene was evident on induction of GST and QR, but not on GSH, G-Rd, or P4501A.