Effect of temperature control upon a mouse model of partial hepatic ischaemia/reperfusion injury.

In vivo models of hepatic ischaemia/reperfusion injury (IRI) are widely used to study both the mechanisms of hepatic ischaemic injury and to seek means of hepatic protection. Achieving high-quality reproducible data are essential if the results of multiple studies are to be compared and reconciled. This paper presents our findings concerning the effect of intraoperative thermoregulation upon signal to noise ratios of hepatic IRI experiments in mice. Four experiments were conducted, using three different strategies for core temperature maintenance. Animals underwent hepatic IRI and euthanized 24 h postoperatively for measurement of plasma alanine aminotransferase (ALT). Duration of ischaemia was used to adjust the severity of injury. Experiment 1 utilized a constant output heating system and resulted in rising postoperative ALTs following increasing durations of hepatic ischaemia. Experiment 2, using the same constant output heating system confirmed a difference between ischaemic and sham-operated animals. Experiment 3 used a thermostatically controlled heating system and resulted in highly variable results with a small, but statistically significant correlation between ALT levels and rectal temperature readings. Experiment 4 used a homeothermic warming system and demonstrated highly reproducible data from increasing durations of ischaemia. High-quality data from hepatic ischaemia/reperfusion models are dependent upon careful control of intraoperative temperature. The use of homeothermic warming systems is recommended and conversely, the use of thermostatically controlled warming mats is to be avoided in these models.

Clinical development of leukocyte cyclooxygenase 2 activity as a systemic biomarker for cancer chemopreventive agents.

Advancement of cancer prevention and therapy requires clinical development of systemic biomarkers of pharmacological efficacy of the agent under scrutiny. Curcumin, a polyphenol derived from Curcuma spp., has shown wide-ranging chemopreventive activity in preclinical carcinogenic models, in which it inhibits cyclooxygenase (COX)-2 at the transcriptional level. COX-2 has been implicated in the development of many human cancers. To explore the inhibition of COX-2 activity as a systemic biomarker of drug efficacy, a biomarker of potential use in clinical trials of many chemopreventive drugs known to inhibit this enzyme, we measured COX-2 protein induction and prostaglandin E(2) (PGE(2)) production in human blood after incubation with lipopolysaccharide (LPS). When 1 microM curcumin was added in vitro to blood from healthy volunteers, LPS-induced COX-2 protein levels and concomitant PGE(2) production were reduced by 24% and 41%, respectively (P < 0.05 by ANOVA). To test whether effects on COX-2 activity could also be measured after oral dosing in humans, we conducted a dose-escalation pilot study of a standardized formulation of Curcuma extract in 15 patients with advanced colorectal cancer. Basal and LPS-mediated PGE(2) production was measured in blood, twice pretreatment and on days 1, 2, 8, and 29 of treatment. Analysis of basal and LPS-induced PGE(2) production during treatment demonstrated a trend toward dose-dependent inhibition (P < 0.005 by regression analysis), but there was no significant difference compared with values from pretreatment time points. Measurement of leukocyte COX-2 activity should be considered in clinical trials of other agents likely to inhibit this isozyme.

Guidelines for the design and statistical analysis of experiments in papers submitted to ATLA.

In vitro experiments need to be well designed and correctly analysed if they are to achieve their full potential to replace the use of animals in research. An "experiment" is a procedure for collecting scientific data in order to answer a hypothesis, or to provide material for generating new hypotheses, and differs from a survey because the scientist has control over the treatments that can be applied. Most experiments can be classified into one of a few formal designs, the most common being completely randomised, and randomised block designs. These are quite common with in vitro experiments, which are often replicated in time. Some experiments involve a single independent (treatment) variable, while other "factorial" designs simultaneously vary two or more independent variables, such as drug treatment and cell line. Factorial designs often provide additional information at little extra cost. Experiments need to be carefully planned to avoid bias, be powerful yet simple, provide for a valid statistical analysis and, in some cases, have a wide range of applicability. Virtually all experiments need some sort of statistical analysis in order to take account of biological variation among the experimental subjects. Parametric methods using the t test or analysis of variance are usually more powerful than non-parametric methods, provided the underlying assumptions of normality of the residuals and equal variances are approximately valid. The statistical analyses of data from a completely randomised design, and from a randomised-block design are demonstrated in Appendices 1 and 2, and methods of determining sample size are discussed in Appendix 3. Appendix 4 gives a checklist for authors submitting papers to ATLA.

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry.

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.

Experimental approaches to the determination of genetic variability.

Toxicology is concerned with the interaction between xenobiotics and biological molecules directly or indirectly coded in the DNA, and can be regarded as a branch of genetics. There is genetic variation in these interactions, which has important implications for risk assessment and because it can be used as a tool in studying toxic mechanisms. The genetics of susceptibility can be studied by forward or reverse genetics. Forward genetics involves working from an observed phenotype such as susceptibility to a particular xenobiotic and identifying the susceptibility genes. Often, this involves mapping and identifying quantitative trait loci, as most toxic responses have a polygenic mode of inheritance. The use of inbred strains is almost essential. Reverse genetics involves starting with a known genetic polymorphism and determining its effects on the response to xenobiotics. Studies of 'knockout' animals are a good example, although there are many naturally occurring polymorphisms that may affect toxic responses. In both cases, care has to be taken to ensure that the genetic background is carefully controlled in any comparison between animals thought to be carrying susceptible and resistant alleles.

Strain differences in haematological response to chloramphenicol succinate in mice: implications for toxicological research.

Much toxicological research continues to be done using genetically undefined "outbred" stocks of mice and rats, although the case for using isogenic strains has been made repeatedly in the literature over a period of more than two decades. Also, very few studies are conducted using more than one strain, with the result that genetic variation in response is seldom apparent to the investigator. Here we report qualitative and quantitative strain differences in the haematological response to chloramphenicol succinate (CAPS) when administered by gavage at 500-2500 mg/kg for 7 days, to four inbred strains of mouse (C3H/He, CBA/Ca, BALB/c and C57BL/6) and one outbred stock (CD-1). CAPS caused anaemia and reticulocytopenia in all mouse strains, and leucopenia in the inbred strains but not in the outbred CD-1 stock. All four inbred strains showed significant (P<0.01) responses to CAPS at lower dose levels than in CD-1 mice, which were phenotypically more variable than the inbred animals. A simulated experiment, using a sample of records from the present study, showed that the use of two mice at each dose level using CD-1, CBA, BALB/c and C57BL/6 (48 total mice), would have given a more sensitive experiment than the use of 47 CD-1 mice alone, and would also have shown that the response is partly strain dependent. These studies provide additional evidence that inbred strains, because of their greater sensitivity and other valuable properties, should be more widely used in toxicology.

A promoter function of the CCCGGG Sma I recognition sequence and its specific role in determining p53 status and identifying DNA damaging agents.

The tumour suppressor p53 protein regulates many genes involved in cellular responses to DNA damage. To date, a common transcriptionally active DNA-binding site for p53 in vivo has not been identified. The pGL3-Basic vector contains a modified fire-fly luciferase cDNA designated luc+ and is designed for studying putative regulatory sequences as it lacks any known eukaryotic promoter sequences. We report here that the CCCGGG sequence, a Sma I site, in the cloning region of the pGL3-Basic vector can promote p53-dependent transcription of the luc+ gene. We have demonstrated, by electrophoretic mobility shift assay (EMSA), that human p53 is able to bind to the CCCGGG sequence in vitro. These data provide the first demonstration that the CCCGGG sequence is a transcriptionally active DNA-binding site for p53. Thus, the pGL3-Basic vector could be used as an indicator of p53 transcriptional activity, to determine the p53 status of cell lines and to identify DNA damaging agents that initiate the activation of p53. The CCCGGG sequence has been found to be present in a number of promoter regions of p53-regulated genes. This and the present study suggest that the CCCGGG sequence may be a consensus sequence recognized by p53 in vivo and may be used to identify genes whose expression may be controlled by p53.

Genealogies of mouse inbred strains.

The mouse is a prime organism of choice for modelling human disease. Over 450 inbred strains of mice have been described, providing a wealth of different genotypes and phenotypes for genetic and other studies. As new strains are generated and others become extinct, it is useful to review periodically what strains are available and how they are related to each other, particularly in the light of available DNA polymorphism data from microsatellite and other markers. We describe the origins and relationships of inbred mouse strains, 90 years after the generation of the first inbred strain. Given the large collection of inbred strains available, and that published information on these strains is incomplete, we propose that all genealogical and genetic data on inbred strains be submitted to a common electronic database to ensure this valuable information resource is preserved and used efficiently.

Reduction in animal use in the production and testing of biologicals.

In the control of biologicals, animals are used largely to measure the concentration of a specific substance, rather than as a "model" of humans, so there is considerable scope for the development of replacement alternatives. When animals continue to be be used, a critical analysis of guidelines and regulations has suggested many ways in which the use of animals could be reduced [1]. However, the widespread failure to use genetically and microbiologically defined animals is scientifically questionable and almost certainly results in the use of excessive numbers. International standardisation on a small number of genetically defined F1 hybrid mice should lead to greater precision in individual tests as well as greater comparability among different laboratories.

Warning: the use of heterogeneous mice may seriously damage your research.

Genetically heterogeneous (GH) mice and rats continue to be widely used in research even though the case for using isogenic strains has been argued repeatedly. The paper by Miller et al. in this issue appears to be the only one in the last 22 to attempt a scientific justification for the continued use of (a limited subset of) GH stocks. However, although they are to be commended for bravery, they fail to make their case. GH stocks represent poor material for controlled studies because genetic heterogeneity normally leads to phenotypic variability and a decline in experimental sensitivity. To counter this argument, Miller et al. claim that phenotypic variability may actually be smaller in GH animals than in their isogenic parents. Were this so (e.g., all mice being short lived, small, and aggressive), it is difficult to see how the use of such a stock could increase the generality of research results based on it, as claimed by Miller et al. Isogenic strains are a vital, proven, and powerful resource for biomedical research, and should be used in preference to GH stocks by all scientists who use laboratory rodents.

Low levels of p53 are associated with resistance to tetrachlorodibenzo-p-dioxin toxicity in DBA/2 mice.

We show here that DBA/2 strain mice have a complex mutation/polymorphism in the promoter region of the Trp53 locus (the mouse p53 locus). This region has previously been shown to be essential for p53 expression. We further show that the DBA/2 mutation is associated with approximately fourfold lower p53 levels in thymocytes treated with the DNA-damaging agent etoposide in-vitro, and with relative resistance of these thymocytes to apoptosis induced by the DNA-damaging agent etoposide compared with C57BL/6 mice. When part of the promoter containing this mutation was inserted into a plasmid containing a luciferase reporter gene but lacking eukaryote promoter sequences and transfected into MCF-7 human breast cell line cells, the mean luciferase activity was slightly less with the DBA/2 than with the C57BL/6 promoter-reporter construct (p < 0.01). We found that DBA/2xC57BL/6 F2 hybrid mice with the DBA/2 genotype at the Trp53 locus were relatively resistant to tetrachlorodibenzo-p-dioxin toxicity, and this resistance was additive with resistance associated with the Ahr locus. DBA/2 mice are long-lived and do not have particularly high levels of cancer, suggesting either that they carry other compensatory tumour resistance alleles (such as Ahr(d)), or that, while there may be a p53 protein dosage effect for acute toxicity, lower than normal levels of p53 may still be sufficient to protect against cancer. In evolutionary terms, it may be better to maintain low levels of p53 in order to avoid death from acute toxicity, even at the expense of a higher incidence of cancer in later life.

Genetic typing of the senescence-accelerated mouse (SAM) strains with microsatellite markers.

The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series.

At least four loci and gender are associated with susceptibility to the chemical induction of lung adenomas in A/J x BALB/c mice.

Four putative quantitative trait loci (QTLs) that influence susceptibility to the induction of lung adenomas by urethane in an F2 cross between A/J and BALB/cOlaHsd have been mapped. Following microsatellite typing of mice with resistant and susceptible phenotypes at 97 microsatellite marker loci, a major locus was identified on chromosome 18 with a lod score of 15. This was responsible for an 8- to 10-fold increase in tumor multiplicity in males and females, respectively, having the AA and CC genotypes at the D18Mit188 marker locus. It mapped close to Dcc (deleted in colorectal cancer). A locus on chromosome 4 (lod score 6.5) had the resistant allele in strain A/J and the susceptible allele in BALB/c, with a 14-fold difference in tumor multiplicity between mice of the AA and CC genotypes. This mapped close to the Cdkn2a (cyclin-dependent kinase inhibitor 2A) locus, which is commonly deleted in mouse lung tumors. Two loci with smaller effects (lod scores 3.03 and 3.25) were identified on chromosomes 1 and 11. There was also significant sexual dimorphism in tumor multiplicity both among 151 F2 hybrids and among 52 mice resulting from a backcross to strain A/J, with males having higher tumor counts than females.

Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene.

A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

Smad4 (homolog of human DPC4) and Smad2 (homolog of human JV18-1): candidates for murine lung tumor resistance and suppressor genes.

In this study we investigated the mouse mad-related genes, Smad4/Dpc4 and Smad2 (homolog of JV18-1), as candidates for involvement in lung tumor resistance and suppression. These genes are located in a region of mouse chromosome 18 that is syntenic with human 18q21.1, where several genes that are mutated in various cancers have been mapped. A newly identified murine lung tumor resistance locus, Par2 has also been mapped to this region of chromosome 18. We found no mutations in the coding regions of either gene in 11 lung tumors from B6CF1 (C57BL/6 x BALB/c) mice by RT-PCR and SSCP/RFLP, suggesting that these genes are not mutated in lung carcinogenesis in this strain. Moreover, loss of heterozygosity in this region of chromosome 18 was not detected in 28 lung adenocarcinomas from B6CF1 mice, 17 lung adenocarcinomas from B6C3F1 mice or 18 lung adenocarcinomas from AB6F1 mice. These data provide evidence that a 'classical' tumor suppressor gene for mouse lung carcinogenesis in these strains does not reside in this region. In order to investigate Smad4/Dpc4 and Smad2 as candidates for the Par2 resistance locus mapped to this region, we also sequenced the coding regions of both genes in cDNA from normal lungs of A/J, BALB/c and C57BL/6 inbred strains of mice. No polymorphisms were detected in the coding region of Smad4. In Smad2, two sequence polymorphisms were identified that are not in the conserved regions of the gene. Northern blot analysis revealed no differential expression in normal lung tissue among the three strains for either gene. Thus, in this study we found no evidence that either Smad4 or Smad2 represents the Par2 lung tumor resistance locus or is a lung tumor suppressor gene in the B6CF1 mice.