Exercise-induced pulmonary haemorrhage during submaximal exercise.

REASONS FOR PERFORMING STUDY: Maximally exercising horses achieve mean pulmonary artery pressures (Ppa(mean)) that exceed the minimum threshold (75 mmHg) estimated for pulmonary capillary rupture and exercise-induced pulmonary haemorrhage (EIPH). EIPH is not expected to occur during moderate submaximal exercise (i.e. 40-60% VO2max) since Ppa(mean) remains well below this threshold. HYPOTHESIS: Prolonged submaximal exercise (trotting) would precipitate locomotory respiratory uncoupling and cause EIPH. This would be present as a result of the most negative intrapleural pressures (as estimated by the minimum oesophageal pressure; Poes(min)) occurring simultaneously with the most positive Ppa (Ppa(peak)) to produce estimated maximal pulmonary artery transmural pressures (PATMPmax) that surpass the EIPH threshold. METHODS: Five Thoroughbred horses trotted to fatigue (approximately 25 min) at 5 m/sec on a 10% incline. Ventilation (V(E)), Poes, and Ppa were measured at 5 min intervals, and bronchoalveolar lavage (BAL) red blood cells (RBCs) were quantified 45 min post exercise. RESULTS: BAL revealed an increased EIPH (rest: 2.0 +/- 1 x 10(5), exercise: 17 +/- 10 x 10(5) RBCs/ml BALF; P<0.05), despite the highest Ppamean reaching only mean +/- s.e. 55 +/- 3 mmHg, while V(E), tidal volume and Poes(min) approached 70-80% of the values achieved at maximal running speeds (10% incline: 12-13 m/sec) by these same horses. The resulting PATMPmax was well above the level considered causative of EIPH. CONCLUSIONS: The finding of significant EIPH during submaximal exercise broadens the spectrum of performance horses susceptible to EIPH and supports studies that suggest that extravascular factors are of primary importance in the aetiology of EIPH. POTENTIAL RELEVANCE: Consideration of strategies such as the equine nasal strip for reducing negative extravascular pressures is warranted even for exercise at moderate intensities.

Effectiveness of an experimental consensus phytase in improving dietary phytate-phosphorus utilization by weanling pigs.

Consensus phytase is a new biosynthetic, heat-stable enzyme derived from the sequences of multiple homologous phytases. Two experiments were conducted to determine its effectiveness, relative to inorganic P and a mutant enzyme of Escherichia coli phytase (Mutant-EP), in improving dietary phytate-P availability to pigs. In Exp. 1, 36 pigs (3 wk old, 7.00 +/- 0.24 kg of BW) were fed a low-P corn-soybean meal basal diet plus consensus phytase at 0, 250, 500, 750, 1,000, or 1,250 U/kg of feed for 5 wk. Plasma inorganic P concentration, plasma alkaline phosphatase activity, bone strength, and overall ADG and gain:feed ratio of pigs were improved (P < 0.05) by consensus phytase in both linear (R2 = 0.20 to 0.70) and quadratic (R2 = 0.30 to 0.70) dose-dependent fashions. In Exp. 2, 36 pigs (4 wk old, 9.61 +/- 0.52 kg BW) were fed the basal diet + inorganic P at 0.1 or 0.2%, consensus phytase at 750 or 450 U/kg of feed, Mutant-EP at 450 U/kg of feed, or 225 U consensus + 225 U Mutant-EP/kg of feed. Pigs fed 750 U of consensus phytase or 450 U of Mutant-EP/kg feed had plasma inorganic concentrations and bone strength that fell between those of pigs fed 0.1 or 0.2% inorganic P. These two measures were 16 to 29% lower (P < 0.05) in pigs fed 450 U of consensus phytase/kg of feed than those of pigs fed 0.2% inorganic P. Plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed Mutant-EP vs. consensus phytase at 450 U/kg at wk 2 and 3. In conclusion, the experimental consensus phytase effectively releases phytate P from the corn-soy diet for weanling pigs. The inorganic P equivalent of 750 U of consensus phytase/kg of feed may fall between 0.1 and 0.2%, but this requires further determination.

Analysis of lacI mutations in Big Blue transgenic mice subjected to parasite-induced inflammation.

Parasite infections have long been associated with specific types of human cancers. Schistosoma hematobium is an inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of hepatocellular carcinoma. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica-induced inflammation in mice to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. This provides a perspective on the relationship between chronic inflammation and cancer and may be a model for future studies on this complex association. In previous studies using the Big Blue transgenic mouse assay, we demonstrated an increase in lacI mutations in liver cells harvested from mice harboring F. hepatica flukes when compared to uninfected control animals. In these studies, we report on the types of mutations associated with this parasite infection. The observed mutational spectrum roughly corresponded to the spectrum of spontaneous mutations in liver cells when compared to control (uninfected) animals. However, the spectrum of mutations from parasitized animals showed a significant increase in complex changes and multiple mutations (18.2%) when compared to what would be expected from control animals (2.8%).

Induced expression of CYP2A5 in inflamed trematode-infested mouse liver.

Trematode infections have long been associated with specific types of cancer. We investigated the ability of the liver fluke Fasciola hepatica to alter host enzymes in a manner that might provide insight into the phenomenon of biologically associated cancers. Our data demonstrate an increased activity of the CYP2A5 isozyme in male mouse liver infected with F.hepatica. Induction of this enzyme was further assessed immunohistochemically. The infection affected CYP2A5 distribution in hepatic tissue. Inflammation and proliferation in liver tissue were observed at the same time that CYP2A5 activity increased. This enzyme is known to participate in the metabolism of several carcinogens which are common contaminants in environments of developing countries where parasitic infections may be prevalent.

Enhanced liver cell mutations in trematode-infected Big Blue transgenic mice.

Parasite infections in humans have long been associated with specific types of cancers. Schistosoma hematobium is a known inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of liver cell cancers. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica as a model agent to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. Using the Big Blue(R) transgenic mouse assay, we found a two-fold increase in lacI mutations in cells harvested from mice harboring F. hepatica worms when compared to uninfected control animals. These data indicate that biological infections can cause increased genetic damage in surrounding host tissue.

Effect of selected antimutagens on the genotoxicity of antitumor agents.

Cyclophosphamide (CP), bleomycin (BL), doxorubicin (DOX) and cisplatin (CISP) are potent antitumor drugs used worldwide against many forms of human cancer. As with most such agents, there can be physiological side-effects and the possible induction of mutations and other genotoxic effects in non-tumor cells. It is common for patients to ingest a host of food supplements to diminish the discomforting side-effects of therapy. Because these food supplements are often also rich in antimutagens that could also affect the biological efficacy of the antitumor drugs, we investigated if such antimutagenic agents were indeed antimutagenic to these antitumor drugs. Using the Salmonella/microsome bioassay, we tested CP, BL, DOX, and CP for mutagenicity in the presence and absence of the antimutagens ascorbic acid (AA), chlorophyllin (CHL) and (+)-catechin (CAT). AA was a very effective antimutagen against CISP and less effective against BL and DOX. It was not antimutagenic to CP. CHL was effective as an antimutagen against all four antitumor drugs, and CAT was a strong inhibitor of DOX mutagenicity, but had little effect on BL, CP and CISP. These data now provide a basis for future in vivo antitumor/antimutagen combination studies to determine if these antimutagens function in a manner to reduce genetic effects without having concomitant effects on intended antitumorogenicity of these therapeutic agents.

In vivo effects of chlorophyllin on the antitumour agent cyclophosphamide.

Cyclophosphamide (CP) is a potent antitumour agent used against many forms of cancer and against certain other diseases. Chlorophyllin (CHL), which is obtained by hydrolysis of chlorophyll to remove phytyl alcohol, is an efficient antimutagenic agent and has been used as a dietary supplement or to diminish the intensity of the discomforting side effects of CP therapy. We undertook to determine the antimutagenic effectiveness of CHL against CP in a mouse model and to determine whether the antitumour efficacy of CP was compromised in vivo by CHL treatment. Experiments utilised CHL administered either in drinking water (1%) for 2 days before treatment, or by gavage (200 mg/kg) 2 hr before treatment with CP (220 mg/kg). Urinary mutagenicity following CP treatment, as determined by the Salmonella/microsome assay, was decreased by both regimes of CHL co-treatment. Similarly, the increase in micronuclei in bone marrow polychromatic erythrocytes in response to CP was reduced by concomitant CHL treatment. In contrast, antitumour efficacy, as determined by growth delay of Colon 38 adenocarcinomas, was not diminished by CHL treatment. We conclude that CHL may have beneficial effects when used in combination with CP therapy.

Mechanism of antimutagenic action of (+)-catechin against the plant-activated aromatic amine 4-nitro-o-phenylenediamine.

Aromatic amines are activated into mutagens by both animal and plant systems. For plant-activated aromatic amines an important step in this process involves peroxidase enzymes. 4-nitro-o-phenylenediamine (NOP) is a well known direct-acting mutagen that can be enhanced in mutagenic potency by intact plant cells and also by isolated peroxidase enzymes. This activation process is inhibited by several different chemical agents including potassium cyanide (KCn), a known peroxidase inhibitor, and (+)-catechin. In our laboratory both KCn and (+)-catechin inhibited peroxidase-mediated NOP activation into a Salmonella mutagen. However, while KCn demonstrated strong peroxidase enzyme inhibition (as measured biochemically), (+)-catechin showed only minimal inhibition of peroxidase. Experiments comparing NOP direct and plant-activated mutagenic activity to different Salmonella strains (in the presence and absence of (+)-catechin) suggest that (+)-catechin may inhibit the mutagenic process by limiting O-acetyltransferase (OAT) activity in Salmonella. OAT activity in Salmonella is a required process for mutations to be induced following treatment with NOP and other aromatic amines.

Characterization of 4-nitro-o-phenylenediamine activation by plant systems.

4-Nitro-o-phenylenediamine (NOP) is a powerful direct-acting mutagen which demonstrates significant enhancement in mutagenicity when exposed to plant enzymatic systems. Evidence implicating the involvement of peroxidactic oxidation in NOP activation has been obtained from plant-cell suspension and isolated enzyme experiments. Using selected cytochrome P450 and peroxidase enzyme inhibitors in conjunction with Salmonella typhimurium strain TA98 and intact plant-cell activating systems as well as isolated horseradish peroxidase enzyme we have further investigated NOP activation by plant systems. The activation of NOP by both plant cells and by horseradish peroxidase was suppressed by the P450 inhibitors methimazole and (+)-catechin and by the peroxidase inhibitors diethyldithiocarbamate and potassium cyanide, but was not suppressed by the P450 inhibitors metyrapone and 7,8-benzoflavone. In addition, peroxidase enzymatic activity was measured and found to be inhibited by methimazole, diethyldithiocarbamate and potassium cyanide but not by (+)-catechin. The data strongly support the involvement of exogenous peroxidase in the plant activation of NOP, but point to a complex metabolic system that requires multistep processing before full mutagenic potential of the plant-activated component of NOP is expressed.

Implications for the involvement of the immune system in parasite-associated cancers.

Biological factors can be carcinogenic risk factors in humans and in animals. Numerous theories have been developed to explain the causal link between biologically-associated disease and the ensuing neoplasia. In this paper we discuss the merits of one of these theories, the possible association between the mammalian inflammatory response and cancer.

Metabolism of 2-aminofluorene by human polymorphonuclear leukocytes: more evidence for the association between inflammation and cancer.

Recent investigations have demonstrated the ability of leukocytes to metabolize promutagens or procarcinogens into their genotoxic forms. As a possible explanation for the association between inflammation and cancer, we and others have hypothesized that local accumulations of leukocytes could take up nearby promutagens, metabolize them, and release genotoxic agents that may cause damage in the surrounding tissue. Using a modified, two-step preincubation protocol with Salmonella, we have tested this hypothesis. We have shown that total human peripheral blood leukocytes, cultured in the presence of 2-aminofluorene for 18 hr, can metabolize 2-aminofluorene into agents mutagenic to Salmonella typhimurium strain TA98. Furthermore, experiments in which polymorphonuclear leukocytes were separated from mononuclear leukocytes demonstrated that the PMNs metabolized 2-aminofluorene to a much greater extent than the MNs.