Derivation of a new lamb survival trait for the New Zealand sheep industry.

Previous research identified that a review of the current industry New Zealand lamb survival trait was necessary as its recording accuracy was reliant on farmers notifying their Sheep Improvement Limited bureau of lamb deaths. This paper reports the decision rules and genetic parameters for a new lamb survival trait for the New Zealand sheep industry. These rules define the new lamb survival trait (NEWSUR) using lamb birth fate (BFATE) codes and the presence/absence of lamb weight measurements. Six univariate animal models were tested and used to estimate variance or covariance components and the resulting direct and maternal heritabilities for NEWSUR. The models differed in the way they adjust for the effect of day of birth, the exclusion or inclusion of a litter (dam/year of birth) random effect, and the application or not of a logit transformation of the phenotypes. For both the linear and logistic methods, models including the random effect of litter provided the best fit for NEWSUR according to log-likelihood values. Log-likelihoods for the linear and logistic models cannot be compared; therefore, a cross-validation method was used to assess whether the logit transformation was appropriate by analyzing the predictive ability of the models. The mean square errors were slightly lower for the linear compared with the logistic model, and therefore, the linear model was recommended for industry use. The heritability attributed to direct effects ranged from 2 to 5.5%. A direct heritability of 5.5% resulted from a linear model without litter effect and omitting the effect of day of birth on survival, whereas a direct heritability of 2% resulted from the logistic model fitting a random litter effect. The heritability attributed to maternal genetic effects ranged from 1.9 to 7.7%. A maternal genetic heritability of 7.7% resulted from the logistic model omitting the litter effect, whereas a maternal genetic heritability of 1.9% resulted from the linear model fitting a random litter effect. The addition of the litter random effect substantially decreased the maternal heritabilities in all cases and was recommended for industry use to avoid overestimation of the maternal genetic variance. Sheep Improvement Limited has implemented NEWSUR and the associated genetic evaluation model based on information described in this paper. Industry-wide implementation will enable sheep breeders to produce more accurate genetic evaluations to their commercial clients.

Genetic parameters for lamb birth weight, survival and death risk traits.

This paper reports genetic parameters for lamb survival and mortality traits on sheep farms in New Zealand. Lamb survival and mortality records were obtained from 38 flocks (103,357 lambs) from 5 yr of lambing data (2007 to 2011) and include many breeds and their crosses (predominantly Romney, Perendale, Coopworth, and Texel). A number of models were tested, all including environmental weather effects and investigating the random environmental effect of dam and litter (dam/year) as well as logit transformation for binary traits. Total heritability (direct + maternal) estimates were low for lamb viability at birth (0.01), lamb death risk to dystocia (0.01), and lamb death risk to starvation exposure (0.01) from birth to 3 d of age in an analysis accounting for direct and maternal genetic effects and the maternal environmental effects. Lamb survival heritabilities reported are very low (total heritabilities range from 0.02 to 0.06). The total heritabilities for the lamb death risk traits are lower than reported estimates of survival to 3 d of age or to weaning suggesting selection for the postmortem traits are not warranted at this time within these flocks. The total heritability for lamb birth weight was moderate (0.38) and the genetic correlations with the lamb death risk traits suggested that directional selection on lamb birth weight would have an effect on survival, although it is likely to have a nonlinear effect and therefore an optimum birth weight at which survival is maximized. This study has also shown that the total heritabilities may be overestimated when not accounting for maternal genetic and environment effects and in particular not accounting for the random environmental effect of litter (dam/year).

Genetic parameters for ewe rearing performance.

This paper reports genetic parameters for ewe performance traits in sheep breeders' flocks in New Zealand. Animal performance records from the AgResearch Lamb Survival Database and from Sheep Improvement Limited were used to generate data sets from 3 lambing years (2003 to 2005) in 24 flocks, and involving 31,651 ewes and many breeds and breed compositions (predominantly Romney, Coopworth, and Texel). The heritabilities and repeatabilities for the litter survival traits were very low. Litter weight traits had heritabilities ranging from 0.12 for BW of lamb weaned to 0.28 for total triplet litter weight at birth and repeatabilities ranging from 0.18 to 0.29. The repeatabilities of BCS and maternal behavior score were low to moderate. This study showed that there is little to be gained from including litter survival in sheep selection programs because heritabilities and repeatabilities for the litter survival traits were very low. However, genetic gains in BCS, maternal behavior score, litter weight at birth, and litter weight weaned are possible in this population. Incorporating these traits into sheep selection programs warrants investigation to improve ewe and therefore flock performance.

Management of maternal-offspring behavior to improve lamb survival in easy care sheep systems.

This paper examines the environmental and management factors affecting lamb survival on high-performing sheep farms in New Zealand. Improved lambing percentage is the biggest contributor to higher profits on New Zealand sheep farms. Many sheep breeders have selected and bred ewes for increased fecundity over the last 4 decades. The increased proportion of ewes having triplets is of concern to farmers and to industry because neonatal lamb mortality is highest in triplets. The majority of lamb deaths occur in the first 3 d after birth and range from 5 to 30% for individual sheep flocks. The ability of a lamb to survive to weaning is determined by genetics, behavior, physiology, and the environment, including on-farm management practices. We investigated the effects of dam body condition in pregnancy, weather during lambing, lamb birth weight, and maternal behavior on single, twin, and triplet lamb viability at birth, lamb death risks from dystocia, and starvation exposure and survival through to weaning for 20 industry flocks from 2003 to 2004 (15,821 lambs). Ewes with higher body condition scores in mid pregnancy had heavier lambs at birth (P < 0.01). Lambs weighing 5.5 to 6 kg at birth (P < 0.01) were more likely to be viable at birth and survive to weaning than heavier or lighter lambs. Weather conditions during late pregnancy (P < 0.05) proved more important than conditions during lambing (P < 0.05) in determining lamb viability and survival through to weaning. Older ewes and ewes with triplets require considerably more attention for farmers to realize their production potential. This information can help formulate appropriate management programs to improve lamb survival rates under easy care farming systems.

A putative autosomal gene increasing ovulation rate in Romney sheep.

Ovulation rates were measured in 547 progeny of 24 rams in a Romney flock with a long history of high prolificacy. These sheep were from the same family line and the distribution of ovulation rates suggests the presence of a segregating major gene (FecW) that increases prolificacy. The phenotype differs from those previously described for major genes affecting prolificacy in sheep. The putative gene shows autosomal inheritance and one copy increases ovulation rate by 0.8-1.0 eggs per ewe ovulating. To date, we have found no evidence of infertility among putative homozygous ewes, as described in some autosomal major genes for prolificacy.

Immunotoxicology assessment in the pharmaceutical industry.

Through several inter-laboratory evaluation studies, selected methods were optimized and evaluated using reference compounds in rodents, to determine their predictive value for detecting toxicity to the immune system. These provide the basis for the OECD, EPA and pending FDA Guidelines. To describe the status of immunotoxicity evaluation using these methods in the pharmaceutical industry, a survey was conducted, and is reported, of ongoing activities among the major pharmaceutical companies. The results describe which assays are performed, how compounds are selected for evaluation; and when, during the drug development process, an evaluation is performed. Finally, the strategy at Sanofi, for the evaluation and application of immunotoxicity methods during the preclinical development of new molecular entities (NMEs) is described. During the past 8 years, Sanofi has evaluated more than 27 NMEs from multiple therapeutic classes as well as four reference compounds (azathioprine, dexamethasone, cyclophosphamide and cyclosporin A). Our experience with multiple animal species (rat, dog and monkey) and immunotoxicity assays selected from the recommended tiers as well as the outcome from the evaluation of our NMEs and reference standards, is described. This experience has led us to believe that immunotoxicology parameters represent an important adjunct for the safety assessment of NMEs. In addition, these methods were easily integrated into the drug development process and yielded an unexpectedly low frequency of positive results. In summary, immunotoxicity can be evaluated on a case-by-case basis driven by pathology or clinical hematology findings, by the drug's indication, the chemical class or indication of the NME evaluated (for example, anti-viral agents), or systematically performed.

Pharmacokinetics of the hypoxic cell cytotoxic agent tirapazamine and its major bioreductive metabolites in mice and humans: retrospective analysis of a pharmacokinetically guided dose-escalation strategy in a phase I trial.

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; SR 259075) is a selective hypoxic cell cytotoxic agent that is bioreductively activated in tumours to a reactive-drug free radical. Preclinically the agent has been shown to possess additive and synergistic anti-tumour activity in combination with radiotherapy and chemotherapy regimens. In the present study the pharmacokinetics and metabolism of tirapazamine were investigated in mice and patients as part of pre-clinical and phase I investigations. The objectives of this work were twofold; firstly, to evaluate retrospectively the utility of a pharmacokinetically guided dose-escalation (PGDE) strategy for tirapazamine, and secondly, to investigate if pharmacologically relevant plasma concentrations could be achieved at tolerable doses. Pharmacokinetic studies for PGDE were conducted in mice at four dose levels ranging from one-tenth of the LD10 to the LD50. The AUC at the LD10 (2932 micrograms ml-1 min) was used to determine a target AUC value of 1173 micrograms ml-1 min (equivalent to 40% of the mouse LD10 AUC) for clinical studies. A phase I study to investigate the tolerance of a single i.v. infusion of tirapazamine (once every 3 weeks) was initiated with close pharmacokinetic monitoring. The starting dose (36 mg/m2) was based on toxicity data obtained in the mouse, rat and dog. Doses were escalated by increases in the volume and duration of infusion. A retrospective analysis of the pharmacokinetic and toxicity data was then made to determine the utility of a PGDE approach. The drug exhibited a steep dose-lethality relationship in mice (LD10 294 mg/m2, LD50 303 mg/m2). The major gross toxicities were body-weight loss (15-20%), pilo-erection and hypoactivity at all dose levels. Sporadic ptosis and conjunctivitis were observed at doses of > 300 mg/m2. The plasma elimination of tirapazamine fitted a monoexponential open model, with rapid elimination from the plasma (t1/2 = 36 +/- 0.65 min) occurring at the LD10 dose of 294 mg/m2. A 10.3-fold increase in dose resulted in a 25.0-fold increase in AUC. Clinically, doses were escalated over the range of 36-450 mg/m2. Ototoxicity (tinnitus and reversible hearing loss) was dose-limiting at 450 mg/m2 and the MTD was 390 mg/m2 for this schedule. Pharmacokinetic analyses in patients revealed that the elimination of tirapazamine in patients was generally bi-phasic, with low inter-patient variability being found in clearance. A 12.5-fold increase in dose resulted in a 19.0-fold increase in AUC. There was good quantitative agreement in metabolite formation between mice and humans with respect to the two- and four-electron bioreductive metabolites. AUC values recorded for tirapazamine at the MTD of 390 mg/m2 (range 1035-1611 micrograms ml-1 min) were similar to the target AUC in mice. Importantly, these levels are consistent with the levels required for radiation-dose enhancement and effective combination with cisplatin in mice. Given (a) the similarities in plasma pharmacokinetics and metabolism observed at the target AUC/MTD in mice, rats, dogs and humans, (b) the similar degree of plasma protein binding seen between species and (c) the relatively low inter-patient variability noted in drug clearance, a successful PGDE approach should have been feasible. The results also indicate that potentially therapeutic levels of tirapazamine are achievable in patients at tolerable doses.

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist, zanoterone.

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6 beta-, 7 alpha-, and 16 alpha-hydroxytestosterone (b beta-, 7 alpha-, and 16 alpha-OHT) and the dealkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes from rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and beta-naphthoflavone (beta-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6 beta-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than the produced by PB or beta-NF (< or = threefold increases). Z (high dose), PB, and beta-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2-2.9-fold in females. beta-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6 beta position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6 beta-OHT suggests that Z induced CYP 3A activity. These findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting, Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily.

The integration of immunotoxicology in drug discovery and development: Investigative and in vitro possibilities.

The development of new pharmaceuticals requires a tremendous investment of time and resources. The early integration of investigative toxicology studies and new toxicological methods, such as immunotoxicological studies, into the selection of new drug candidates facilities compound safety evaluation, reduces risk, and improves development cycle time. Experience at Sterling Winthrop Inc. has led us to believe that immunotoxicology assessment represents an important and useful tool in drug discovery and development, but that it should not be applied as a routine screening procedure. Instead, immunotoxicity evaluation should be initiated as a response to knowledge about the class of drug under investigation, or when data from other studies suggest an effect on the immune system. Three examples of this approach are given and each one demonstrates the potential of in vitro immunotoxicology assays to reduce the time, resources and animal use required in the discovery and development of new drug candidates.

DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships.

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which possess carcinogenic as well as anticarcinogenic activity in vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC greater than ISC); riddelline (DPC greater than ISC); retrorsine (DPC greater than ISC); senecionine (DPC greater than ISC); heliosupine (DPC greater than ISC); monocrotaline (ISC = DPC); latifoline (DPC greater than ISC); and retronecine (ISC greater than DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were greater than 90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an alpha,beta-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on biological activity.

Mechanisms of toxicity of hepsulfam in human tumor cell lines.

1,7-Heptanediol disulfamate (hepsulfam, NSC 329680) is a new anti-cancer agent which is currently undergoing phase I clinical trials. The mechanism of action of this compound is not clear at this time. We have recently shown that hepsulfam was more toxic to L1210 leukemia cells than was busulfan. Consistent with the difference in toxicity, we found that hepsulfam induced DNA interstrand cross-links in L1210 mouse leukemia cells, whereas busulfan did not. In the present study, we have found that hepsulfam was more cytotoxic to two human leukemia cell lines (HL-60 and K562) and to two human colon carcinoma cell lines (BE and HT-29) than was busulfan. As in L1210 cells, hepsulfam induced a higher level of DNA interstrand cross-links than busulfan. Both compounds induced DNA-protein cross-links. Hepsulfam was also more cytotoxic to the human leukemia cell lines when the concentrations were reduced 10-fold and the duration of drug exposure was increased to 12-h This more accurately reflects the drug exposures that human leukemia cells may encounter in vivo. Under these 12-h drug exposures, hepsulfam was still able to form DNA interstrand and DNA-protein cross-links, whereas busulfan was only able to form DNA-protein cross-links. These results show that busulfan and hepsulfam react with DNA differently and that hepsulfam is a more potent cytotoxic agent.

In vitro studies on the mechanism of action of hepsulfam in chronic myelogenous leukemia patients.

In the present study we have characterized the cytotoxicity and DNA damage induced by hepsulfam and busulfan in cells isolated from both chronic myelogenous leukemia (CML) patients and normal donors. hepsulfam inhibited colony-forming units-granulocyte, macrophage to a greater extent than busulfan in peripheral blood cells (PBCs) isolated from CML patients. Normal PBCs were equally sensitive to both agents and were more sensitive than the cells isolated from CML patients. Hepsulfam induced DNA interstrand cross-links in PBCs and bone marrow from both CML and normal volunteers, whereas busulfan produced few or no DNA interstrand cross-links. In addition, hepsulfam induced higher levels of DNA interstrand cross-linking than busulfam in three samples isolated from CML patients in blast crisis. Busulfan did however cause a small number of DNA strand breaks to be formed in human cells. Both agents produced similar levels of DNA-protein cross-links in PBCs from CML patients. These results suggest that the mechanism of DNA reactivity of hepsulfam and busulfan differ and that hepsulfam may prove useful in the treatment of CML.

Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution.

Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN2), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents.

Effects of phenytoin and carbamazepine on human natural killer cell activity and genotoxicity in vitro.

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers and exposed in vitro to phenytoin or carbamazepine, two widely used antiepileptic drugs (AED). This study investigated the effects of these drugs on natural killer (NK) cell activity and antibody-dependent cell-mediated cytotoxicity (ADCC), which are both thought to protect against developing neoplasms. Also, the genotoxicity of phenytoin on human PBMC was investigated by gravity-flow alkaline elution. Concentrations of phenytoin considered therapeutic (10 and 20 micrograms/ml) and a dose considered acutely toxic (40 micrograms/ml) were used while carbamazepine levels of 8 micrograms/ml (therapeutic) and 10 and 16 micrograms/ml (acutely toxic) were tested. Phenytoin at all three concentrations significantly suppressed NK cell activity in a dose-dependent manner. Carbamazepine had no significant effect on NK cell activity at the dose levels studied. Incubation in propylene glycol, the diluent for carbamazepine, significantly decreased NK cell activity compared to saline. Phenytoin also significantly depressed interferon augmentation of NK cell cytotoxicity in a dose dependent manner. ADCC activity was significantly depressed with 20 and 40 micrograms/ml phenytoin. Alkaline elution showed a slight but significant increase in DNA single-strand breaks of PBMC exposed to 40 micrograms/ml phenytoin for 18 or 72 hr. These results show phenytoin may induce pronounced immunosuppression of NK cell and ADCC activity in patients receiving antiepileptic therapy and that this agent has a potential for genotoxic side effects. Phenytoin may also increase the potential for neoplasm development by a direct interaction with cellular DNA and/or an indirect mechanism by immunosuppression.

Effects of inducer pretreatment on liver function and morphology in the mountain vole Microtus montanus.

Liver function and morphology of the mountain vole, Microtus montanus, were examined after i.p. injections of phenobarbital, beta-naphthoflavone, or Aroclor 1254 at three dose levels. The results of the liver function tests showed serum glutamic pyruvic transaminase and serum malathion carboxylesterase activities were normal in all the treatment groups. The histological results showed no necrotic tissue but did reveal two different morphological stages related to the level of monooxygenase activity; a low induction state was represented by foamy vacuolated hepatocytes while high induction states were related to enlarged, swollen, hypertrophied cells.

Gravity-flow alkaline elution: a method to rapidly detect carcinogen-induced DNA strand breaks.

A rapid, sensitive and reliable gravity-flow alkaline elution assay was developed to detect DNA strand breaks in cultured Madin-Darby bovine kidney epithelial cells. Elution was completed within 2 h without the use of pumps. The system was validated by exposing the cells to X-irradiation (25-1500 R) which resulted in a significant dose dependent response (p less than 0.05) with excellent correlation (r-0.93). The assay reliably detected the DNA damage of seven genotoxic carcinogens. In general, the measured DNA damage was dose dependent and significantly different from control values for all genotoxic carcinogens tested. Six non-genotoxic compounds were tested and showed no detectable DNA damage.

Effects of varying inducer type and dose on hepatic monooxygenase activities in the mountain vole Microtus montanus.

Hepatic monooxygenase activities of the mountain vole, Microtus montanus, were measured after i.p. injections of phenobarbital, B-naphthoflavone and Aroclor 1254 at doses ranging from 5 to 80 mg/kg. The results showed that mountain voles differed in their induction of hepatic monooxygenase activity relative to other rodents. The results also suggest substrate specificity in the detection of enzymatic induction and the importance of considering effects of varying inducer doses on hepatic monooxygenases.