The acute effects of rumen pulse-dosing of hydrogen acceptors during methane inhibition with nitrate or 3-nitrooxypropanol in dairy cows.

Dietary methane (CH4) mitigation is in some cases associated with an increased hydrogen (H2) emission. The objective of the present study was to investigate the acute and short-term effects of acceptors for H2 (fumaric acid, acrylic acid, or phloroglucinol) supplemented via pulse-dosing to dairy cows fed CH4 mitigating diets (using nitrate or 3-nitrooxypropanol), on gas exchange, rumen gas, and VFA composition. For this purpose, 2 individual 4 × 4 Latin square experiments were conducted with 4 periods of 3 d (nitrate supplementation) and 7 d (3-nitrooxypropanol supplementation), respectively. In each study, 4 rumen-cannulated Danish Holstein cows were used. Each additive for CH4 mitigation was included in the ad libitum-fed diet within the 2 experiments (exp. 1 and exp. 2), to which the cows were adapted for at least 14 d. Acceptors for H2 were administered twice daily in equal portions through the rumen fistula immediately after feeding of the individual cow. In exp. 1 (nitrate), the treatments were CON-1 (no H2-acceptor), FUM-1 (fumaric acid), ACR-1 (acrylic acid), and FUM+ACR-1 (50% FUM-1 + 50% ACR-1). In exp. 2 (3-nitrooxypropanol), the 3 treatments, CON-2, FUM-2, and ACR-2, were similar to CON-1, FUM-1 and ACR-1 treatments, however the fourth treatment was PHL-2 (phloroglucinol). Gas exchanges were measured in respiration chambers, and samples of rumen liquid and headspace gas were taken in time series relative to feeding and dosing on specific days. Headspace gas was analyzed for gas composition, and rumen liquid was analyzed for VFA composition and dissolved gas concentrations. Headspace gas composition and dissolved gas concentration were only measured in exp. 2. Dry matter intake was reduced upon acrylic acid supplementation. There were no significant effects of any treatments in any experiments on H2 emission, except for a decrease in hourly H2 emission rate (g/h) at 1 h after feeding in both experiments. In exp. 2, H2 headspace proportions increased with ACR-2 supplementation, whereas dissolved concentrations were unaffected. In exp. 1, cows on ACR-1 increased propionate proportion at 1 h after feeding. In exp. 2, both FUM-2 and ACR-2 increased rumen propionate proportion in the hours after feeding and dosing. There was no effect on rumen acetate for cows on PHL-2. There was a strong positive correlation between rumen dissolved CH4 and headspace CH4 (r = 0.84), whereas the equivalent correlation was weaker for H2 (r = 0.41). For the relationship between dissolved concentrations and emissions of CH4 and H2, there was a moderate positive correlation for CH4 (r = 0.54), whereas it was weak for H2 (r = 0.28) with zero slope. In conclusion, the results suggested that fumaric acid and acrylic acid to some extent was reduced to propionate without associative effects on measures for H2 redirection. Furthermore, phloroglucinol seemed not to be metabolized in the rumen in the present study, because no effects on rumen acetate or measures of H2 were observed. Changes in H2 headspace and emission may be a poor proxy for actual changes in the rumen fluid concentration of H2.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.

Enteric methane emission, milk production, and composition of dairy cows fed 3-nitrooxypropanol.

This study examined the effect of 3-nitrooxypropanol (3-NOP), an investigational substance, on enteric methane emission, milk production, and composition in Holstein dairy cows. Following a 3-wk covariate period, 48 multi- and primiparous cows averaging (± standard deviation) 118 ± 28 d in milk, 43.4 ± 8 kg/d milk yield, and 594 ± 57 kg of body weight were blocked based on days in milk, milk yield, and enteric methane emission and randomly assigned to 1 of 2 treatment groups: (1) control, no 3-NOP, and (2) 3-NOP applied at 60 mg/kg feed dry matter. Inclusion of 3-NOP was through the total mixed ration and fed for 15 consecutive weeks. Cows were housed in a freestall barn equipped with a Calan Broadbent Feeding System (American Calan Inc., Northwood, NH) for monitoring individual dry matter intake and fed ad libitum once daily. Enteric gaseous emissions (methane, carbon dioxide, and hydrogen) were measured using 3 GreenFeed (C-Lock Inc., Rapid City, SD) units. Dry matter intake, cow body weight, and body weight change were not affected by 3-NOP. Compared with the control group, 3-NOP applied at 60 mg/kg feed dry matter decreased daily methane emission, emission yield, and emission intensity by 26, 27, and 29%, respectively. Enteric emission of carbon dioxide was not affected, and hydrogen emission was increased 6-fold by 3-NOP. Administration of 3-NOP had no effect on milk and energy-corrected milk yields and feed efficiency, increased milk fat and milk urea nitrogen concentrations, and increased milk fat yield but had no other effects on milk components. Concentration of C6:0 and C8:0 and the sum of saturated fatty acids in milk fat were increased by 3-NOP. Total trans fatty acids and the sum of polyunsaturated fatty acids were decreased by 3-NOP. In this experiment, 3-NOP decreased enteric methane daily emission, yield, and intensity without affecting dry matter intake and milk yield, but increased milk fat in high-producing dairy cows.

Effects of 3-nitrooxypropanol and varying concentrate feed proportions in the ration on methane emission, rumen fermentation and performance of periparturient dairy cows.

The climate-relevant enteric methane (CH4) formation represents a loss of feed energy that is potentially meaningful for energetically undersupplied peripartal dairy cows. Higher concentrate feed proportions (CFP) are known to reduce CH4 emissions in cows. The same applies to the feed additive 3-nitrooxypropanol (3-NOP), albeit through different mechanisms. It was hypothesised that the hydrogen not utilised for CH4 formation through the inhibition by 3-NOP would be sequestered by propionate formation triggered by higher CFP so that it could thereby give rise to a synergistically reduced CH4 emission. In a 2 × 2-factorial design, low (LC) or high (HC) CFP were either tested without supplements (CONLC, CONHC) or combined with 3-NOP (NOPLC, 48.4 mg/kg dry matter (DM); NOPHC, 51.2 mg 3-NOP/kg DM). These four rations were fed to a total of 55 Holstein cows from d 28 ante partum until d 120 post partum. DM intake (DMI) was not affected by 3-NOP but increased with CFP (CFP; p < 0.001). CH4/DMI and CH4/energy-corrected milk (ECM) were mitigated by 3-NOP (23% NOPLC, 33% NOPHC) (p < 0.001) and high CFP (12% CON, 22% 3-NOP groups) (CFP × TIME p < 0.001). Under the conditions of the present experiment, the CH4 emissions of NOPLC increased to the level of the CON groups from week 8 until the end of trial (3-NOP × CFP × TIME; p < 0.01). CO2 yield decreased by 3-NOP and high CFP (3-NOP × CFP; p < 0.001). The reduced body weight loss and feed efficiency in HC groups paralleled a more positive energy balance being most obvious in NOPHC (3-NOP × CFP; p < 0.001). ECM was lower for NOPHC compared to CONHC (3-NOP × CFP; p < 0.05), whereas LC groups did not differ. A decreased fat to protein ratio was observed in HC groups and, until week 6 post partum, in NOPLC. Milk lactose and urea increased by 3-NOP (3-NOP; p < 0.05). 3-NOP and high CFP changed rumen fermentation to a more propionic-metabolic profile (3-NOP; CFP; p < 0.01) but did not affect rumen pH. In conclusion, CH4 emission was synergistically reduced when high CFP was combined with 3-NOP while the CH4 mitigating 3-NOP effect decreased with progressing time when the supplement was added to the high-forage ration. The nature of these interactions needs to be clarified.

Reducing enteric methane emissions from dairy cattle: Two ways to supplement 3-nitrooxypropanol.

The aim of this work was to determine the effect of 3-nitrooxypropanol (3-NOP) on the enteric methane (CH4) emissions and performance of lactating dairy cows when mixed in with roughage or incorporated into a concentrate pellet. After 2 pretreatment weeks without 3-NOP supplementation, 30 Holstein Friesian cows were divided into 3 homogeneous treatment groups: no additive, 3-NOP mixed in with the basal diet (roughage; NOPbas), and 3-NOP incorporated into a concentrate pellet (NOPconc). The pretreatment period was followed by a 10-wk treatment period in which the NOPbas and NOPconc cows were fed 1.6 g of 3-NOP/cow per day. After the treatment period, a 2-wk washout period followed without 3-NOP supplementation. The CH4 emissions were measured using a GreenFeed unit (C-Lock Inc., Rapid City, SD) installed in a freestall with cubicles during the entire experimental period. On average for the total treatment period and compared with the no-additive group, CH4 production (g/d) was 28 and 23% lower for NOPbas and NOPconc, respectively. Methane yield (g/kg of dry matter intake) and methane intensity (g/kg of milk) were 23 and 24% lower for NOPbas, respectively, and 21 and 22% lower for NOPconc, respectively. No differences were found between NOPbas and NOPconc. Moreover, supplying 3-NOP did not affect total dry matter intake, milk production, or milk composition. The results of this experiment show that 3-NOP can reduce enteric CH4 emissions of dairy cattle when incorporated into a concentrate pellet and that this reduction is not different from the effect of mixing in 3-NOP with the basal diet (roughage). This broadens the possibilities for using 3-NOP in the dairy sector worldwide, as it is not always feasible to provide an additive mixed in with the basal diet.

Can the epoxides of cinnamyl alcohol and cinnamal show new cases of contact allergy?

BACKGROUND: Cinnamyl alcohol is considered to be a prohapten and prehapten with cinnamal as the main metabolite. However, many individuals who are allergic to cinnamyl alcohol do not react to cinnamal. Sensitizing epoxides of cinnamyl alcohol and cinnamal have been identified as metabolites and autoxidation products of cinnamyl alcohol. OBJECTIVE: To investigate the clinical relevance of contact allergy to epoxycinnamyl alcohol and epoxycinnamal. METHODS: Irritative effects of the epoxides were investigated in 12 dermatitis patients. Epoxycinnamyl alcohol and epoxycinnamal were patch tested in 393 and 390 consecutive patients, respectively. In parallel, cinnamyl alcohol and cinnamal were patch tested in 607 and 616 patients, respectively. RESULTS: Both epoxides were irritants, but no more positive reactions were detected than when testing was performed with cinnamyl alcohol and cinnamal. Late allergic reactions to epoxycinnamyl alcohol were observed. In general, patients with late reactions showed doubtful or positive reactions to cinnamal and fragrance mix I at regular patch testing. CONCLUSION: The investigated epoxides are not important haptens in contact allergy to cinnamon fragrance. The high frequency of fragrance allergy among patients included in the irritancy study showed the difficulty of suspecting fragrance allergy on the basis of history; patch testing broadly with fragrance compounds is therefore important.

Orally administered glycidol and its fatty acid esters as well as 3-MCPD fatty acid esters are metabolized to 3-MCPD in the F344 rat.

IARC has classified glycidol and 3-monochloropropane-1,2-diol (3-MCPD) as group 2A and 2B, respectively. Their esters are generated in foodstuffs during processing and there are concerns that they may be hydrolyzed to the carcinogenic forms in vivo. Thus, we conducted two studies. In the first, we administered glycidol and 3-MCPD and associated esters (glycidol oleate: GO, glycidol linoleate: GL, 3-MCPD dipalmitate: CDP, 3-MCPD monopalmitate: CMP, 3-MCPD dioleate: CDO) to male F344 rats by single oral gavage. After 30 min, 3-MCPD was detected in serum from all groups. Glycidol was detected in serum from the rats given glycidol or GL and CDP and CDO in serum from rats given these compounds. In the second, we examined if metabolism occurs on simple reaction with rat intestinal contents (gastric, duodenal and cecal contents) from male F344 gpt delta rats. Newly produced 3-MCPD was detected in all gut contents incubated with the three 3-MCPD fatty acid esters and in gastric and duodenal contents incubated with glycidol and in duodenal and cecal contents incubated with GO. Although our observation was performed at 1 time point, the results showed that not only 3-MCPD esters but also glycidol and glycidol esters are metabolized into 3-MCPD in the rat.

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose.

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75mg/kg bw, and diHOPrVal levels were measured 24h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R(2)=0.943). Blood sampling at different time points (1, 10, 20, or 40days) from four groups administered glycidol at 12mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61days), with the calculated first-order elimination rate constant (kel) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (kval) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The kval was 6.7±1.1 and 5.6±1.3 (pmol/g globin per µMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from kval and diHOPrVal levels were in agreement with the area under the (concentration-time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment.

Effect of combining the methanogenesis inhibitor 3-nitrooxypropanol and cottonseeds on methane emissions, feed intake, and milk production of grazing dairy cows.

No single enteric CH4 mitigating strategy has been consistently effective or is readily applicable to ruminants in grassland systems. When CH4 mitigating strategies are effective under grazing conditions, mitigation is mild to moderate at best. A study was conducted to evaluate the potential of combining two CH4 mitigation strategies deemed feasible to apply in grazing dairy cows, the methanogenesis inhibitor 3-nitrooxypropanol additive (3-NOP) and cottonseed supplementation (CTS), seeking to enhance their individual CH4 mitigating potential. Forty-eight dairy cows were evaluated in a continuous grazing study and supplemented with either a starch-based concentrate (STA) or one that contained cottonseeds (1.75 kg DM/d; CTS), and with either 19 g/d of 10% 3-NOP (Bovaer®) or the additive's carrier (placebo), in a 2 × 2 factorial arrangement of treatments. Treatments were supplied mixed with a concentrate supplement (5 kg/d as fed) and offered in two equal rations at milking. Methane emissions were measured on weeks 4 and 8 using the sulphur hexafluoride tracer gas technique over a 5-d period. The 3-NOP and CTS treatments tended to interact on absolute CH4 such that 3-NOP decreased CH4 by 13.4% with STA, but there was no mitigation with 3-NOP and CTS. Treatment interactions were also obtained for CH4 yield, where 3-NOP tended to decrease CH4 when supplied with STA, and tended to increase it with CTS. The increase in CH4 yield with the CTS diet was driven by a numerical decrease in DM intake. Methane intensity was not affected by the 3-NOP or CTS treatments. Total volatile fatty acids in ruminal fluid were not affected by 3-NOP supplementation, but a reduction in acetate and an increase in propionate proportion occurred, resulting in decreased acetate: propionate. The 3-NOP additive decreased grass intake; however, energy-corrected milk yield and milk composition were largely unaffected. Milk urea increased with 3-NOP supplementation. Combining twice daily supplementation of 3-NOP and CTS did not enhance their CH4 mitigation potential when fed to grazing dairy cows. The relatively low inhibition of CH4 production by 3-NOP compared to studies with total mixed rations may result from the mode of delivery (pulse dosed twice daily) and time gap caused by experimental handling and moving of animals to pasture after 3-NOP supplementation in the milking parlour, which could have impaired the synchrony between the additive presence in the rumen and grass intake in paddocks.

Relative oral bioavailability of glycidol from glycidyl fatty acid esters in rats.

In order to quantify the relative bioavailability of glycidol from glycidyl fatty acid esters in vivo, glycidyl palmitoyl ester and glycidol were orally applied to rats in equimolar doses. The time courses of the amounts of glycidol binding to hemoglobin as well as the excretion of 2,3-dihydroxypropyl mercapturic acids were determined. The results indicate that glycidol is released from the glycidyl ester by hydrolysis and rapidly distributed in the organism. In relation to glycidol, there was only a small timely delay in the binding to hemoglobin for the glycidol moiety released from the ester which may be certainly attributed to enzymatic hydrolysis. In both cases, however, an analogous plateau was observed representing similar amounts of hemoglobin binding. With regard to the urinary excretion of mercapturic acids, also similar amounts of dihydroxypropyl mercapturic acids could be detected. In an ADME test using a virtual double tag (³H, ¹4C) of glycidyl palmitoyl ester, a diverging isotope distribution was detected. The kinetics of the ¹4C-activity reflected the kinetics of free glycidol released after hydrolysis of the palmitoyl ester. In view of this experimental data obtained in rats, it is at present justified for the purpose of risk assessment to assume complete hydrolysis of the glycidyl ester in the gastrointestinal tract. Therefore, assessment of human exposure to glycidyl fatty acid ester should be regarded as an exposure to the same molar quantity of glycidol.

Investigation of activity of monoterpenes and phenylpropanoids against immature stages of Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae).

The objective of this study was to assess the acaricidal activity of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde on unengorged larvae and nymphs of Amblyomma cajennense and Rhipicephalus sanguineus, using the modified larval packet test. Carvacrol, eugenol, and (E)-cinnamaldehyde were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, while thymol was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml, in all cases with 10 repetitions per treatment. For the A. cajennense larvae, mortality rates caused by carvacrol, thymol, eugenol, and (E)-cinnamaldehyde at the lowest concentration were 45.0, 62.7, 10.2, and 81.6%, respectively, reached 100% at the concentration of 5.0 µl/ml for carvacrol and (E)-cinnamaldehyde and 5.0 mg/ml for thymol, while this mortality was observed at 15.0 µl/ml for eugenol. For the nymphs of this species, carvacrol and thymol caused 100% mortality starting at a concentration of 5.0 µl/ml and 10.0 mg/ml, respectively, while eugenol caused 100% mortality at 20.0 µl/ml and the mortality caused by (E)-cinnamaldehyde did not exceed 64%. In the tests with R. sanguineus larvae, the lowest concentration of carvacrol and (E)-cinnamaldehyde resulted in 100% mortality, while this percentage was observed starting at 10.0 µl/ml for eugenol. For nymphs, carvacrol and thymol at the smallest concentration caused 100% lethality, unlike the results for eugenol and (E)-cinnamaldehyde, where 100% mortality was only observed starting at the concentration of 10.0 µl/ml. The results obtained indicate that the tested substances have acaricidal activity on unengorged larvae and nymphs of A. cajennense and R. sanguineus.

Discovery of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P(1) receptor agonist with high selectivity against S1P(3) and enhanced efficacy in lowering peripheral lymphocyte counts in mice.

Urinary Excretion of 2/3-Monochloropropanediol (2/3-MCPD) and 2,3-Dihydroxypropylmercapturic Acid (DHPMA) after a Single High dose of Fatty Acid Esters of 2/3-MCPD and Glycidol: A Controlled Exposure Study in Humans.

SCOPE: 2- and 3-monochloropropanediol (2/3-MCPD) and glycidol are absorbed in the intestine after lipase-catalyzed hydrolysis of their fatty acid esters. METHODS AND RESULTS: In an exposure study with 12 non-smoking participants, the complete urinary excretion of the metabolite 2,3-dihydroxypropylmercapturic acid (DHPMA) and of 2/3-MCPD is measured on four consecutive days before and after consumption of 50 g glycidyl ester-rich palm fat or 12 g 2/3-MCPD ester-rich hazelnut oil. After controlled exposure, urinary excretion rates of 2/3-MCPD per hour strongly increase, followed by a decrease with average half-lives of 5.8 h (2-MCPD) and 3.6 h (3-MCPD). After consumption of hazelnut oil, mean excretion rates are 14.3% (2-MCPD) and 3.7% (3-MCPD) of the study doses. The latter rate is significantly higher (4.6%) after consumption of palm fat, indicating partial conversion (about 5%) of glycidol to 3-MCPD under the acidic conditions in the stomach. The average daily "background" exposure is estimated to be 0.12 and 0.32 µg per kg body weight (BW) for 2-MCPD and 3-MCPD, respectively. The relatively high and constant urinary excretion of DHPMA does not reflect the controlled exposure. CONCLUSION: Urinary excretion of 2- and 3-MCPD is suitable as biomarker for the external exposure to the respective fatty acid esters.

Greenhouse gas and ammonia emissions from stored manure from beef cattle supplemented 3-nitrooxypropanol and monensin to reduce enteric methane emissions.

The investigative material 3-nitrooxypropanol (3-NOP) can reduce enteric methane emissions from beef cattle. North American beef cattle are often supplemented the drug monensin to improve feed digestibility. Residual and confounding effects of these additives on manure greenhouse gas (GHG) emissions are unknown. This research tested whether manure carbon and nitrogen, and GHG and ammonia emissions, differed from cattle fed a typical finishing diet and 3-NOP [125-200 mg kg-1 dry matter (DM) feed], or both 3-NOP (125-200 mg kg-1 DM) and monensin (33 mg kg-1 DM) together, compared to a control (no supplements) when manure was stockpiled or composted for 202 days. Consistent with other studies, cumulative GHGs (except nitrous oxide) and ammonia emissions were higher from composted compared to stockpiled manure (all P < 0.01). Dry matter, total carbon and total nitrogen mass balance estimates, and cumulative GHG and ammonia emissions, from stored manure were not affected by 3-NOP or monensin. During the current experiment, supplementing beef cattle with 3-NOP did not significantly affect manure GHG or NH3 emissions during storage under the tested management conditions, suggesting supplementing cattle with 3-NOP does not have residual effects on manure decomposition as estimated using total carbon and nitrogen losses and GHG emissions.

Evaluation of nitro compounds as feed additives in diets of Eimeria-challenged broilers in vitro and in vivo.

Coccidiosis is a disease caused by Eimeria spp., resulting in approximately 3 billion US dollar loss in the poultry industry annually. The present study evaluated the effects of potential feed additives, 2-Nitro-1-propanol (NP) and nitroethanol (NE), on control of coccidiosis. An in vitro experiment indicated that both NP and NE inhibited the development of sporozoites in Madin-Darby bovine kidney cells (MDBK). The in vivo study was further conducted to evaluate the effects of NP and NE on growth performance, nitrogen-corrected apparent metabolizable energy (AMEn), and intestinal lesion scores of broilers challenged with Eimeria spps. Six treatments were tested in the study, including the nonchallenged control, challenged control, 100 ppm NP, 200 ppm NP, 100 ppm NE, and 200 ppm NE. Broilers were fed the treatment diets from day 12 until the end of the trial. All birds except the unchallenged control were challenged with Eimeria maxima, Eimeria tenella, and Eimeria acervulina on day 14. The growth performance was calculated, and the intestinal lesion was scored on day 20. The results showed that Eimeria challenge significantly reduced growth performance, increased intestinal lesion scores, and decreased AMEn compared with the nonchallenged control group. Birds fed with 200 ppm of NP had reduced growth performance compared with the nonchallenged control and challenged control. However, the supplementation of NP significantly improved AMEn and reduced cecal damage. Overall, NP and NE reduced sporozoites numbers in the MDBK cells. NP improved dietary digestibility of energy and reduces lesion scores in the ceca but could not maintain growth performance in broiler chickens infected with Eimeria spp.

Net reductions in greenhouse gas emissions from feed additive use in California dairy cattle.

The livestock industry is one of the main contributors to greenhouse gas emissions and there is an increasing demand for the industry to reduce its carbon footprint. Several studies have shown that feed additives 3-nitroxypropanol and nitrate to be effective in reducing enteric methane emissions. The objective of this study was to estimate the net mitigating effect of using 3-nitroxypropanol and nitrate on total greenhouse gas emissions in California dairy industry. A life cycle assessment approach was used to conduct a cradle-to-farm gate environmental impact analysis based on dairy production system in California. Emissions associated with crop production, feed additive production, enteric methane, farm management, and manure storage were calculated and expressed as kg CO2 equivalents (CO2e) per kg of energy corrected milk. The total greenhouse gas emissions from baseline, 3-nitroxypropanol and nitrate offered during lactation were 1.12, 0.993, and 1.08 kg CO2e/kg energy corrected milk, respectively. The average net reduction rates for 3-nitroxypropanol and nitrate were 11.7% and 3.95%, respectively. In both cases, using the feed additives on the whole herd slightly improved overall carbon footprint reduction compared to limiting its use during lactation phase. Although both 3-nitroxypropanol and nitrate had effects on decreasing the total greenhouse gas emission, the former was much more effective with no known safety issues in reducing the carbon footprint of dairy production in California.

Effect of allyl alcohol on hepatic transporter expression: zonal patterns of expression and role of Kupffer cell function.

During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.

Cancer risk estimation of glycidol based on rodent carcinogenicity studies, a multiplicative risk model and in vivo dosimetry.

Here we evaluate a multiplicative (relative) risk model for improved cancer risk estimation of genotoxic compounds. According to this model, cancer risk is proportional to the background tumor incidence and to the internal dose of the genotoxic compound. Furthermore, the relative risk coefficient per internal dose is considered to be approximately the same across tumor sites, sex, and species. In the present study, we demonstrate that the relative risk model is valid for cancer risk estimation of glycidol, a common food contaminant. Published tumor data from glycidol carcinogenicity studies in mice and rats were evaluated in combination with internal dose estimates from hemoglobin adduct measurements in blood from mice and rats treated with glycidol in short-term studies. A good agreement between predicted and observed tumor incidence in responding sites was demonstrated in the animals, supporting a relative risk coefficient that is independent of tumor site, sex, and species. There was no significant difference between the risk coefficients for mice (5.1% per mMh) and rats (5.4% per mMh) when considering internal doses of glycidol. Altogether, this mechanism-based risk model gives a reliable risk coefficient, which then was extrapolated to humans considering internal dose, and background cancer incidence.

A comparative 90-day toxicity study of allyl acetate, allyl alcohol and acrolein.

Allyl acetate (AAC), allyl alcohol (AAL), and acrolein (ACR) are used in the manufacture of detergents, plastics, pharmaceuticals, and chemicals and as agricultural agents. A metabolic relationship exists between these chemicals in which allyl acetate is metabolized to allyl alcohol and subsequently to the highly reactive, alpha,beta-unsaturated aldehyde, acrolein. Due to the weaker reactivity of the protoxicants, allyl acetate and allyl alcohol, relative to acrolien we hypothesized the protoxicants would attain greater systemic exposure and therefore deliver higher doses of acrolein to the internal organs. By extension, the higher systemic exposure to acrolein we hypothesized should lead to more internal organ toxicity in the allyl acetate and allyl alcohol treated animals relative to those treated with acrolein. To address our hypothesis we compared the range of toxicities produced by all three chemicals in male and female Fischer 344/N rats and B6C3F1 mice exposed 5 days a week for 3 months by gavage in 0.5% methylcellulose. Rats (10/group) were dosed with 0-100mg/kg allyl acetate, 0-25mg/kg allyl alcohol, or 0-10mg/kg acrolein. Mice (10/group) were dosed with 0-125mg/kg allyl acetate, 0-50mg/kg allyl alcohol, or 0-20mg/kg acrolein. The highest dose of allyl acetate and acrolein decreased survival in both mice and rats. The primary target organ for the toxicity of all three chemicals in both species and sexes was the forestomach; squamous epithelial hyperplasia was observed following exposure to each chemical. In both species the highest allyl acetate dose group exhibited forestomach epithelium necrosis and hemorrhage and the highest dose of acrolein led to glandular stomach hemorrhage. Liver histopathology was the most apparent with allyl acetate, was also observed with allyl alcohol, but was not observed with acrolein. All chemicals had effects on the hematopoietic system with allyl acetate having the most pronounced effect. When dosed at quantities limited by toxicity, allyl acetate and allyl alcohol produce higher levels of urinary mercapturic acids than the minimally toxic dose of acrolein. This observation is likely due to biotransformation of allyl acetate and ally alcohol to acrolein that occurs after absorption and suggests that these chemicals are protoxicants that increase systemic exposure of acrolein. Increased systemic exposure to acrolein is likely responsible for the differences in hepatic toxicological profile observed with these chemicals.

ClC chloride channels in tooth germ and odontoblast-like MDPC-23 cells.

OBJECTIVE: To detect expression of ClC chloride channel mRNA in tooth germ and odontoblasts, and explore the affect of chloride channel function on cell proliferation and cell cycle. DESIGN: We extracted total RNA of tooth germ from newborn C57BL mice and mouse odontoblast-like cells (MDPC-23), then detected mRNA expression of chloride channel genes Clcn1-7 with RT-PCR. We used chloride channel blocker 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB) to interfere with chloride channel function of MDPC-23 cells. Cell proliferation rate and cell cycle were detected with MTT assay and flow cytometry, respectively. Student's t-test was used to determine statistical significance between control and treatment groups. RESULTS: The mRNA of Clcn1-7 chloride channel genes was expressed in tooth germ of newborn mice. Clcn3, Clcn5 and Clcn7 mRNAs were expressed in MDPC-23 cells. NPPB slowed down the proliferation rate of MDPC-23 cells from day 2 to day 4 (P<0.01), and also changed the proportion of cell cycle phase. Comparing to the control, the proportion of G2/M phase cells reduced from 3.93+/-2.62% to 0.54+/-0.25% (P<0.05). The ratio of G1/G2 increased from 1.86+/-0.01 to 1.95+/-0.02 (P<0.05). CONCLUSIONS: There is abundant chloride channel gene expression in tooth germ. Some of these chloride channels may regulate tooth development through effects on cell proliferation and cell cycle signal pathway.