An improved method to estimate ultrasonic absorption in agar-based gel phantom using thermocouples and MR thermometry.

In this paper, a novel experimental set-up was developed that measures the absorption coefficient. The proposed system was evaluated in an agar-based gel phantom. The new experimental system provides accurate and fast measurement of the rate of temperature change within the phantom. The rate of temperature change was measured using thermocouple and was confirmed using MR thermometry. An ultrasonic transducer with a broad beam was used in order to reduce the conduction effect. The absorption coefficient of the agar-based phantom was 0.26 dB/cm-MHz using 4% agar, 30% evaporated milk and 4% silica. The absorption coefficient increased by increasing the volume of the evaporated milk, and agar. The absorption coefficient increased at low silica concentration (<4%) and then decreased at higher concentration of silica (>4%). By proper selection of evaporated milk, agar and silica concentration, it is possible to achieve similar coefficient like in soft tissues. Acoustic absorption measurement is considered as a difficult measurement in ultrasonics because obtaining the precise temperature change in the focus is challenging. Due to the quick and accurate placement of the thermocouple at the ultrasonic beam, it is possible with the proposed system to perform absorption measurement is less than one minute.

Tissue differences in the modulation of rat cytochromes P450 and phase II conjugation systems by dietary doses of phenethyl isothiocyanate.

Rats were fed diets supplemented with phenethyl isothiocyanate (PEITC) at 0.06 (low dose, dietary intake level), 0.6 (medium dose) and 6.0 micromole/g (high dose), and xenobiotic-metabolising enzymes were monitored in liver, lung and kidney. At the low dose, inhibition of the hepatic O-dealkylation of ethoxy- and methoxyresorufin was noted, whereas at the high dose increases in the O-depentylation of pentoxyresorufin and O-debenzylation of benzyloxyquinoline were observed, whereas p-nitrophenol hydroxylase was inhibited. Hepatic bioactivation of 2-amino-3-methylimidazo-[4,5-f]quinoline to mutagens was not influenced by the PEITC-treatment. In the lung, at the high dose, ethoxyresorufin dealkylation was elevated and that of pentoxyresorufin suppressed; no significant changes were seen in the kidney. Quinone reductase was markedly elevated at all doses in liver, but the lung enzyme was refractive whereas in the kidney a modest rise was observed at the high dose. Hepatic glutathione S-transferase activity was stimulated by PEITC-treatment, but no effect was evident in the lung or kidney. It is concluded that the effects of PEITC on xenobiotic-metabolising systems are dose- and tissue-dependent, with the liver being the most sensitive and the lung generally resistant. Increased detoxication rather than cytochrome P450 inhibition is the likely mechanism of the chemopreventive activity of PEITC.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model.

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Functional integrity of precision-cut liver slices from deer and cattle.

Precision-cut bovine and cervine liver slices were incubated in RPMI 1640 media for up to 72 h, and cellular integrity was assessed utilizing the leakage of lactate dehydrogenase (LDH) and the formation of the formazan metabolite of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Leakage of LDH (80%) from the bovine and cervine slices was noted only following 10 h of culture, and similarly, the generation of MTT-formazan declined. Metabolic viability was determined using 7-ethoxycoumarin as the model substrate, which was metabolized by slices from both animal species in a time-dependent manner for at least 6 h to generate 7-hydroxycoumarin, which was secreted into the media primarily as glucuronide and sulphate conjugates. With both cervine and bovine slices metabolic activity decreased markedly after a 4-h preincubation as assessed following a further 2-h incubation in the presence of 7-ethoxycoumarin. Subsequently, ethoxycoumarin metabolism by bovine slices did not decrease further until 24 h and was still detectable at 72 h. In the case of cervine slices, activity declined gradually after 8 h, with no activity being detectable at 72 h. It may be concluded that cervine and bovine slices may be maintained metabolically active for 8-10 h, which should prove sufficient for xenobiotic metabolism studies to be performed.

Short-term black tea intake modulates the excretion of urinary mutagens in rats treated with 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ): role of CYP1A2 upregulation.

Rats were exposed to black tea (2.5% w/v) as their sole drinking liquid for either 1 day or 1 month, while controls were maintained on water. After this treatment period, all animals received a single oral dose IQ (2-amino-3-methylimidazo-[4,5-f]quinoline), and urine was collected for 48 h. Mutagenic activity of the urine was determined in the Ames test in the presence and absence of an activation system. The excretion of direct-acting mutagens was markedly reduced following tea intake, and was more pronounced after the 1-day treatment. Similarly, both tea treatments suppressed the excretion of indirect-acting mutagens. Furthermore, both tea treatments induced hepatic CYP1A2 activity and expression, but cytosolic glutathione S-transferase activity was only modestly induced in the group of animals receiving tea for 1 day, and only when DCNB (1,2-dichloro-4-nitrobenzene) was used as substrate; glucuronosyl activity was elevated modestly only in the animals receiving the tea for a month. It is concluded that even short-term exposure to black tea is capable of influencing the metabolic fate of IQ, and this is most likely related to the upregulation of CYP1A2.

Metabolic and structural viability of precision-cut rat lung slices in culture.

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h. 2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with beta-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 microm. 3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation. 4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24h; longer incubations did not result in further leakage. 5. It is concluded that precision-cut rat lung slices, of 600 microm thickness, can be maintained metabolically viable in culture for some 8 h.

Bioavailability of dietary doses of 3H-labelled tea antioxidants (+)-catechin and (-)-epicatechin in rat.

1. The bioavailability and pharmacokinetic characteristics of the tea antioxidants (+)-catechin and (-)-epicatechin were investigated in the rat following intake of dietary doses. 2. To achieve this objective, tritiated derivatives (tritium was incorporated at the 3-position of the heterocyclic ring) of these compounds were administered to rats orally and intravenously at dose levels equivalent to human dietary levels of intake. 3. Following intravenous administration of both compounds, about one-third of the dose was excreted in the urine and two-thirds in the faeces, indicating extensive biliary excretion. When the same doses were administered orally, only about 5% of the dose of each compound was recovered in the urine. 4. Comparison of the areas under the curve following oral and intravenous administration revealed that the bioavailability of both compounds was less than 5%. 5. Exchange of tritium with water in the blood occurred 3 h after oral, but not after intravenous, administration of the flavanols to rat. This is believed to represent microbial degradation of the compounds by the gut flora. 6. It was established that the bioavailability of the tea antioxidants (+)-catechin and (-)-epicatechin in the rat following intake of dietary doses was poor.

Hepatic and intestinal cytochrome P450 and conjugase activities in rats treated with black tea theafulvins and theaflavins.

Theaflavins and theafulvins, a fraction of thearubigins, were isolated from aqueous infusions of black tea, and their effects on the hepatic and intestinal cytochrome P450 system, and on the glutathione S-transferase, epoxide hydrolase, glucuronosyl transferase and sulphotransferase enzyme systems were investigated in rats following oral intake for four weeks. Neither theafulvins nor theaflavins influenced cytochrome P450 activity in the liver as exemplified by the O-dealkylations of methoxy-, ethoxy- and pentoxyresorufin, the hydroxylations of lauric acid and p-nitrophenol, and the N-demethylation of erythromycin; similarly, hepatic xenobiotic conjugation systems were unaffected. In the intestine, both polyphenolic fractions markedly suppressed the O-deethylation of ethoxyresorufin and this was accompanied by a decrease in the CYP1A1 apoprotein levels. Probing intestinal microsomes with antibodies to CYP2E1 revealed the presence of a single band in the cytochrome P450 region whose intensity was lower in the polyphenol-treated animals. Immunoblot analysis utilising antibodies to CYP3A showed that the treatment with theafulvins and theaflavins reduced the apoprotein levels. A single band in the cytochrome P450 region was evident when the intestinal microsomes were probed with antibodies to CYP4A1 but the level of expression was not affected by the treatment with the black tea polyphenols. Finally, treatment of the rats with theaflavins had no effect on any of the intestinal conjugating enzymes studied, but treatment with theafulvins led to inhibition of glucuronosyl transferase activity.

Pharmacokinetic interactions between herbal remedies and medicinal drugs.

1. The use of herbal products to treat a wide range of conditions is rising rapidly, leading to increased intake of phytochemicals. Recent studies revealed potentially fatal interactions between herbal remedies and traditional drugs. 2. In transplant patients, self-medication with St John's wort (Hypericum perforatum) has led to a drop in plasma levels of the immunosuppressant drug cyclosporine, causing tissue rejection. 3. Intake of St John's wort increases the expression of intestinal P-glycoprotein and the expression of CYP3A4 in the liver and intestine. The combined up-regulation in intestinal P-glycoprotein and hepatic and intestinal CYP3A4 impairs the absorption and stimulates the metabolism of cyclosporine, leading to subtherapeutic plasma levels. The St John's wort component, hyperforin, contributes to the induction of CYP3A4. 4. St John's wort also enhances the metabolism of other CYP3A4 substrates including the protease inhibitors indinavir and nevirapine, oral contraceptives, and tricyclic antidepressants such as amitriptyline. 5. Other herbal remedies with the potential to modulate cytochrome P450 activity and thus participate in interactions with conventional drugs include Milk thistle, Angelica dahurica, ginseng, garlic preparations, Danshen and liquorice. 6. Herbal products are currently not subject to the rigorous testing indispensable for conventional drugs. However, if potential drug interactions are to be predicted, it is essential that the ability of herbal products to interfere with drug-metabolizing enzyme systems is fully established.

Contribution of CYP1A2 in the hepatic metabolism of melatonin: studies with isolated microsomal preparations and liver slices.

The objective of the present studies was to define the enzyme systems catalysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulphatoxymelatonin in rat hepatic postmitochondrial preparations and in precision-cut liver slices. Melatonin 6-hydroxylase activity was localized in microsomes and was supported by NADPH, but not NADH. Treatment of rats with beta-naphthoflavone more than tripled 6-sulphatoxymelatonin formation from melatonin, but gave rise only to a moderate increase (25%) in the sulphate conjugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, acetone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin generation when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline inhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin without any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with beta-naphthoflavone, and to a lesser extent phenobarbitone, elevated the levels of 6-sulphatoxymelatonin in the culture medium. No such increase was seen when slices from beta-naphthoflavone-treated rats were incubated with 6-hydroxymelatonin, whereas a modest increase was seen with slices from phenobarbitone-treated rats. Treatment of rats with acetone, dexamethasone or clofibrate failed to modulate the levels of 6-sulphatoxymelatonin generated from either melatonin or 6-hydroxymelatonin. Molecular modelling analysis revealed that melatonin had a high area/depth(2) ratio, displayed characteristics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, all the above data provide strong experimental evidence that CYP1A2 is an important catalyst of the 6-hydroxylation of melatonin.

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Mutagenicity of bay-region amino-substituted cyclopenta[a]phenanthrenes and 2- and 5-aminochrysene.

The relative mutagenic potentials of 11-amino-16,17-dihydro-15H-cyclopenta[a]phenanthrene, its 17-keto derivative, and 2- and 5-aminochrysene have been compared in Salmonella typhimurium TA98 and TA100 in the presence of a postmitochondrial liver preparation from Aroclor 1254 induced rats. The 11-amino hydrocarbon is a very weak mutagen (0.27 revertants/nmol), whereas the 11-amino-17-ketone is much more active (129 revertants/nmol). 2-Aminochrysene is the most mutagenic arylamine ( approximately 500 revertants/nmol) among these compounds, but its 5-amino isomer is much less active (0.9 revertants/nmol). Possible reasons for these marked differences are suggested. Use of TA98 with over-expressing O-acetyltransferase (YG 1024) and deficient in this enzyme (TA98/l,8-DNP(6)) with the 11-amino-17-ketone and with 5-aminochrysene clearly indicates the importance of this enzyme in their bioactivation, implying oxidation of the amino group to the hydroxylamine in both these compounds.

The metabolism and bioactivation of agaritine and of other mushroom hydrazines by whole mushroom homogenate and by mushroom tyrosinase.

Whole homogenates of Agaricus bisporus metabolised the mushroom hydrazine agaritine [beta-N-(gamma-L(+)glutamyl)-4-(hydroxymethyl) phenylhydrazine] to generate at least three metabolites. None of these metabolites, however, was the free hydrazine [4-(hydroxymethyl)phenylhydrazine], the postulated metabolite of agaritine believed to be formed as a result of the loss of the gamma-glutamyl group, the reaction being catalysed by gamma-glutamyltransferase. The three metabolites of agaritine displayed weak mutagenic activity towards Salmonella typhimurium strain TA104. 4-(Hydroxymethyl)phenylhydrazine, as the N'-acetyl derivative, was metabolised by mushroom tyrosinase to yield a number of metabolites that induced a mutagenic response in S. typhimurium TA104. Similar to N'-acetyl-4-(hydroxymethyl)phenylhydrazine, agaritine was extensively metabolised by the mushroom tyrosinase but, in contrast, the structurally related N'-acetyl-4-hydrazinobenzoic acid did not serve as substrate of this enzyme, implying a critical role for the hydroxymethyl group at the para-position. In conclusion, the current studies have demonstrated for the first time that: (a) whole mushroom homogenates readily metabolise agaritine but not to the postulated 4-(hydroxymethyl)phenylhydrazine; and (b) mushroom tyrosinase metabolises agaritine and N'-acetyl-4-(hydroxymethyl)phenylhydrazine, in the latter case forming genotoxic metabolites.

Accelerated HER-2 degradation enhances ovarian tumor recognition by CTL. Implications for tumor immunogenicity.

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369-377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.

Effect of antioxidant flavonoids and a food mutagen on lymphocytes of a thalassemia patient without chelation therapy in the Comet assay.

Thalassemia remains a significant health problem in Europe, the Middle East, and Asia. In such patients, generally high iron levels make free oxygen radicals accessible, for example, through Fenton-type chemistry, and generate superoxide and hydroxyl radicals. Increased oxygen radical capacity is known to be associated with cancer and ageing. It was shown in previous studies that peripheral blood lymphocytes from a sickle/beta thal double heterozygote-sickle phenotype, thalassemia patient, not yet on chelation therapy, were more sensitive to the effects of oxygen radicals and iron salts than lymphocytes from normal controls. Iron overload in thalassemia patients can result from dietary absorption. It was considered that with other dietary agents, such as food mutagens and flavonoids, the thalassemia patient might also show increased sensitivity to the effects of these agents. The present study, therefore, compared the effects of the food mutagen/carcinogen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2), in fresh or frozen normal human peripheral lymphocytes with frozen lymphocytes from the same thalassemia patient. The lymphocytes from the thalassemia patient showed an approximately two-fold increase in sensitivity. When a combination of Tryp-P-2, with either quercitin or kaempferol, was compared in frozen lymphocytes and lymphocytes from the thalassemia patient, a two-fold increase in sensitivity was also maintained. Responses to Trp-P-2 were reduced to untreated control levels at the highest doses of quercitin and kaempferol, and were highly significantly different by comparison with Trp-P-2 alone (P<0.001). The flavonoids acted in an antigenotoxic/antioxidant manner. Sensitivity was slightly increased with kaempferol by comparison with quercitin. At low concentrations of the flavonoids there was some evidence of an exacerbation of response, perhaps due to a switch to pro-oxidant status. This exacerbation of response at low doses of flavonoids has been seen in earlier studies with normal lymphocytes. Teratogenesis Carcinog. Mutagen. 21:165-174, 2001.

Peptide priming of cytolytic activity to HER-2 epitope 369-377 in healthy individuals.

The presence of tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients demonstrates an immune response against tumors that apparently cannot control disease spread. This raises concerns as to whether amplification of this response may be useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, may be therapeutically important, based on the hypothesis that CTLs that recognize tumors early may be more effective in containing their progression than CTLs that expand only when the disease progresses. To address the feasibility of priming cytolytic activity in healthy donors, we used the HER-2 peptide E75 (369-377) as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen-presenting cells. We found that of 10 healthy donors tested, two responded at priming with E75 presented on autologous dendritic cells by induction of E75-specific CTL activity. Three other responders were identified after two additional restimulations. Of these five responders, three recognized E75 presented on the ovarian tumor line SKOV3.A2, as demonstrated by cold-target inhibition experiments. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and interleukin 12 but not in the nonresponders. AlphaB7.1 monoclonal antibody added at priming enhanced induction of lytic activity in only one of the four nonresponding donors tested, suggesting that in the majority of donors, E75-precursor CTLs were not tolerized. Because of the possibility that disease may develop in nonresponders, strategies to improve the immunogenicity of tumor antigens for healthy donors may be required for development of cancer vaccines.

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides.

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776-789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884-899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon gamma secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination.

Stability of cytochrome P450 proteins in cultured precision-cut rat liver slices.

The objective of this study was to evaluate the stability of individual, xenobiotic-metabolising, cytochrome P450 proteins in precision-cut rat liver slices cultured for up to 72 h using the multiwell plate system. This was achieved using established diagnostic probes (O-dealkylation of methoxy-, ethoxy- and pentoxy-resorufin, testosterone 2alpha-hydroxylase, debrisoquine 4-hydroxylase, aniline p-hydroxylase and lauric acid hydroxylase) and immunologically using Western blotting. All cytochrome P450 activities declined in culture, the most rapid loss occurring at about 8-12 h of culture; in all cases no detectable activity was present in the 72-h cultured slices. Isoform-specific differences in the stability of various cytochrome P450 proteins were observed, with CYP2E1 being the most stable. When cytochrome P450 expression was determined immunologically, a different picture emerged. High levels of apoprotein were retained in the slices even when activity was very low. In the case of CYP2B, apoprotein levels even increased following the culture of hepatic slices. It is concluded, that for tissue slices to become an acceptable in vitro alternative system for long-term incubations, the culturing conditions must be improved to ensure that cytochrome P450 activities are better maintained.

Oxidant-induced S-glutathiolation inactivates protein kinase C-alpha (PKC-alpha): a potential mechanism of PKC isozyme regulation.

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.

Fate of the mushroom hydrazine agaritine in the rat and mouse.

The fate of the mushroom hydrazine [14C]agaritine was investigated in the mouse and rat strains previously employed in carcinogenicity studies with the edible mushroom Agaricus bisporus. Agaritine was rapidly absorbed in both species, achieving higher blood levels in the mouse, but with similar area under the curve. Covalent binding of agaritine material to proteins was detected only in the liver and kidney, but the extent of binding was the same in the rat and mouse. Most of the radioactivity was excreted during the first 24 hours in both animal species: in the rat it was distributed equally between urine and feces, whereas in the mouse more of the radioactivity was excreted in the urine. No qualitative differences in the metabolic profile were evident, but quantitative differences were observed. Treatment of the urine with deconjugating enzymes did not reveal the presence of any conjugates. Agaritine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzene diazonium ion were not detected in the urine or in the plasma of either species. No mutagens or promutagens were detected by the Ames mutagenicity assay in the urine of either species after exposure to agaritine. Repeated administration of agaritine to rats and mice did not alter the urinary metabolic profile and excretion of radioactivity. Similarly, feeding mice a raw mushroom diet, according to the protocol employed in the carcinogenicity studies, did not modulate the excretion of radioactivity or the urinary metabolic pattern. No major species differences in the fate of agaritine in rat and mouse were noted that could provide a rationale for the carcinogenicity of A. bisporus in the mouse, but not in the rat.