Selective genotyping with epistasis can be utilized for a major quantitative trait locus mapping in hypertension in rats.

Epistasis used to be considered an obstacle in mapping quantitative trait loci (QTL) despite its significance. Numerous epistases have proved to be involved in quantitative genetics. We established a backcross model that demonstrates a major QTL for hypertension (Ht). Seventy-eight backcrossed rats (BC), derived from spontaneously hypertensive rats (SHR) and normotensive Fischer 344 rats, showed bimodal distribution of systolic blood pressure (BP) values and a phenotypic segregation ratio consistent with 1:1. In this backcross analysis, sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (Serca) II heterozygotes showed widespread bimodality in frequency distribution of BP values and obviously demonstrated Ht. First, in genome-wide screening, Mapmaker/QTL analysis mapped Ht at a locus between D1Mgh8 and D1Mit4 near Sa in all 78 BC. The peak logarithm of the odds (LOD) score reached 5.3. Second, Serca II heterozygous and homozygous BC were analyzed separately using Mapmaker/QTL. In the 35 Serca II heterozygous BC, the peak LOD score was 3.8 at the same locus whereas it did not reach statistical significance in the 43 Serca II homozygotes. Third, to map Ht efficiently, we selected 18 Serca II heterozygous BC with 9 highest and 9 lowest BP values. In these 18 BC, the peak LOD score reached 8.1. In 17 of the 18, D1Mgh8 genotypes (homo or hetero) qualitatively cosegregated with BP phenotypes (high or low) (P < 0.0001, by chi-square analysis). In conclusion, selective genotyping with epistasis can be utilized for a major QTL mapping near Sa on chromosome 1 in SHR.

Heart size in inbred strains of rats. Part 1. Genetic determination of the development of cardiovascular enlargement in rats.

The heart and aorta weights in 23 strains of rats and the four-way cross generation among the M520/N, SHRSP/N, SHR/N, and WKY/N strains were investigated in relation to their blood pressure in an attempt to characterize cardiovascular enlargement (increased weight of heart and aorta) from a genetic aspect. The distribution of blood pressure in these strains at 10 weeks of age was clearly divided into hypertensive and normotensive groups. In the hypertensive group, heart weight increased in proportion to blood pressure. In contrast, there was no relationship between blood pressure and heart weight in the normotensive group in spite of large strain differences in heart weights. The result of variance analysis exhibited a significant strain difference in heart weight, and the degree of genetic determination was estimated to be 65%-75%. A similar genetic influence was apparent for normotensive strains excluding hypertensive strains. The distribution of blood pressures in the four-way cross generation showed the segregation of three phenotypes consisting of normotensive, intermediate and hypertensive groups. A large variability was seen in heart weight of each group. However, the increase in average heart weight of these three groups was very small. The degree of genetic determination from the cross analysis was estimated to be 45%-65%. These results indicate that heart weight is a highly heritable trait, and that the effect of genetic factors on cardiac enlargement is larger than that of blood pressure. A similar result was obtained for the aorta weight. However, the effect of genetic factors was less important for aorta weight than for heart weight since the degree of genetic determination was estimated to be 45%-65% from the strain comparison and 35%-60% from the cross analysis.

Heart size in inbred strains of rats. Part 2. Cardiovascular DNA and RNA contents during the development of cardiac enlargement in rats.

Enlargement and nucleic acid content of the cardiovascular system of several strains (SHRSP/N, SHR/N, OM/N, M520/N) of rats were compared with the WKY/N strain in an attempt to characterize cardiac enlargement. Cardiac enlargement in rats can be due to either hypertrophy (increase in myocyte size), hyperplasia (increase in cell number including supporting tissue), or a combination of both. The sum of the indices of the degree of hypertrophy and hyperplasia calculated from the difference of the heart and aorta deoxyribonucleic acid (DNA) concentration and total DNA content between each strain and the WKY/N was almost equal to the degree of heart and aorta enlargement. The SHRSP/N revealed a striking hypertrophy of myocardial cells from the prehypertensive stage, and hyperplasia appeared gradually with the elevation of blood pressure. In contrast, the SHR/N developed a marked hyperplasia with some hypertrophy at the prehypertensive stage. Cardiac enlargement of the OM/N was attributed to both hypertrophy and hyperplasia. A large heart weight of the M520/N was recognized at only a young age, and was due almost entirely to hyperplasia. Aortic enlargements were related to hyperplasia. An increased ribonucleic acid (RNA) concentration was observed in both ventricles of the SHRSP/N, SHR/N, and M520/N rats at 4 weeks of age, and in all of the four strains at 16 weeks of age. A significantly higher RNA concentration was indicated in the aorta of three hypertensive strains of SHRSP/N, SHR/N, and OM/N at established hypertensive stage. These changes might be related to manifestations of genetic or other factors such as the effect of elevated blood pressure.

Possible role of nutritional factors in the incidence of cerebral lesions in stroke-prone spontaneously hypertensive rats.

The incidence of cerebral lesions in stroke-prone spontaneously hypertensive rats appears to depend on the severity of the hypertension and nutritional factors. Comparison of American and Japanese commercial rat diets revealed a much higher incidence of stroke in rats receiving the Japanese diet (88% vs 30% by 9 months of age). Analyses of the diets indicate that perhaps the most important difference in the two diets is the protein content. Based on complete amino acid analyses of the protein in these diets, it appears that the American diet contains about 22% protein as compared to about 15% for the Japanese diet. Minor differences in vitamin and mineral contents are not remarkable. Comparison of the findings in this experimental rat model with epidemiologic studies suggest that nutritional factors may also play a role in the incidence of stroke in humans.

Phospholipase C-delta gene of the spontaneously hypertensive rat harbors point mutations causing amino acid substitutions in a catalytic domain.

This study was undertaken in order to investigate the newly discovered spontaneously hypertensive rat (SHR)-specific restriction fragment length polymorphism (RFLP) at the genomic locus of (poly)phosphoinositide-specific phospholipase C (PLC)-delta at a DNA sequence level. Our aim was to clone the PLC-delta complimentary DNA (cDNA) from SHR and analyse the genomic DNA obtained from two hypertensive rat strains such as SHR and its stroke-prone substrain (SHR-SP) and three normotensive rat strains such as Sprague-Dawley, Donryu and Wistar-Kyoto (WKY) by preparing an aortic cDNA library of SHR, hybridization cloning of PLC-delta cDNA and an analysis of the genomic DNA by polymerase chain reaction. By digesting with restriction enzyme XhoI, we discovered an RFLP band displaying only in SHR and SHR-SP, not in Sprague-Dawley, Donryu and WKY rats. DNA sequencing of PLC-delta cDNA cloned from an aortic cDNA library of SHR revealed a total of three SHR-specific point mutations, two of which resulted in amino acid substitutions. The first point mutation (A to T) was detected at the XhoI site, changing a threonine(ACG) to a serine(TCG), and the second point mutation (A to G) was discovered in the vicinity of the first one, changing an isoleucine(ATA) to a methionine(ATG). This is the first demonstration of the mutations in the SHR genome changing amino acid sequences. These amino acid substitutions, situated in the putative catalytic X domain of PLC-delta, may be the major cause of the augmented PLC activity observed in the SHR, possibly leading to hypertension-related phenonemoma such as abnormal calcium homeostasis and increased intracellular calcium ion concentrations.

Genotypes of sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase II gene in substrains of spontaneously hypertensive rats.

OBJECTIVE: To search for a genetic marker of the sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (SERCA) II gene in spontaneously hypertensive rats (SHRs) and to investigate differences in blood pressure and intracellular Ca2+ among some substrains of SHRs and Wistar-Kyoto (WKY) rats related to their SERCA II genotypes. DESIGN AND METHODS: The coding region of the SERCA II gene was sequenced in SHRs. Blood pressure and intracellular Ca2+ concentration ([Ca2+]i) in platelets were measured in substrains of SHRs and WKY rats with different SERCA II genotypes. RESULTS: A point mutation that provided restriction fragment length polymorphisms (RFLPs) by HindIII or Saul was found in the SERCA II gene. The polymerase chain reaction (PCR) products were digested by HindIII in SHR substrains and WKY-Kyoto rats, whereas they were digested by Saul in normotensive strains and SHR-Toho. Among SHR-Kyoto, SHR-Toho, WKY-Kyoto and WKY-Charles River, the substrains with the HindIII-digested SERCA II genotype showed slightly but significantly higher systolic blood pressure and augmented agonist-stimulated [Ca2+]i than those with the Saul-digested genotype. CONCLUSIONS: RFLPs were found in the SERCA II gene. In the substrain analysis of SHRs and WKY rats, higher blood pressure and increased [Ca2+]i were associated with the SERCA II genotype digested by HindIII. The SERCA II gene locus has the potential to contribute to the development of hypertension and abnormal intracellular Ca2+ metabolism in SHRs. These RFLPs in the SERCA II gene should be a useful genetic marker.

Inherited primary hypothyroidism with thyrotrophin resistance in Japanese cats.

Mutant cats were developed with non-goitrous primary hypothyroidism. They were clinically characterized by severely retarded growth, mild anaemia and high mortality in the young. They responded markedly to thyroid hormone replacement. Thyroid glands in the mutants were normal in position but slightly reduced in size. Laboratory studies revealed low serum concentrations of thyroxine (T4) and tri-iodothyronine (T3), and increased serum concentrations of TSH. Administration of TRH induced no further increase in TSH. Administration of exogenous TSH after suppression of endogenous TSH by T3 did not increase the serum concentration of T4 in the mutants, in sharp contrast with the threefold increase in serum T4 observed in the normal litter-mates. These findings suggest that the underlying pathogenesis of this disorder is unresponsive to TSH. Moreover, we found that the mutants were transmitted in an autosomal recessive manner.

Increased intracellular Ca2+ is not coinherited with an inferred major gene locus for hypertension (ht) in the spontaneously hypertensive rat.

Hypertension is characterized by a complex mode of inheritance, consisting of the accumulation and interaction of major and minor genes. The existence of a single major gene locus (ht) has been demonstrated in the backcross analysis of spontaneously hypertensive rats (SHR) and normotensive Donryu rats. Intracellular Ca2+ concentration ([Ca2+]i) determines the tonus of vascular smooth muscle. It has been hypothesized that abnormal Ca2+ transport is an inheritable trait with profound influence on the development of hypertension. Backcross analysis between SHR and Donryu rats was performed to demonstrate ht and to dissect polygenic hypertensive traits through ht and abnormal intracellular Ca2+ metabolism. Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2+]i in platelets were significantly greater in SHR than in Donryu and F1 rats. The backcrossed rats were distributed into two clusters on a scattergram of blood pressure versus [Ca2+]i, demonstrating the existence of ht. The blood pressure level was correlated with thrombin-stimulated [Ca2+]i in each cluster. Increased [Ca2+]i in platelets was not coinherited with ht and was considered to be a minor inheritable hypertensive trait discriminated from ht. Therefore, [Ca2+]i in platelets is an inadequate marker for searching ht.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 23). A comparative 2- and 4-week repeated oral dose testicular toxicity study of boric acid in rats.

To assess the validity and limitations of 2-week repeated daily dosing to detect toxic effects on male reproductive organs in rodents, a comparative 2- and 4-week oral repeated dosing study of boric acid, a known testicular toxicant, was given to 6- or 8-week-old Crj:Wistar rats at daily levels of 0, 125, 250 and 500 mg/kg. The ages of the rats were selected so that they were all sacrificed at 10 weeks of age. The testes and epididymides were weighed at necropsy; histopathological specimens were prepared in a routine manner and stained with H&E or PAS-H. In addition, the sperm number and motility rates were evaluated. There were no boric acid-induced effects on reproductive organ weights and on gross behavior/appearance in any groups in either the 2- or 4-week studies. The sperm number and motility rate were not decreased in any group after 2 weeks, while both decreased in the 250 and 500 mg/kg groups after 4 weeks. Histopathologically, as evidence of toxicity at the early stage of boric acid exposure, retention of step 19 spermatids of stages IX-XI was observed in the testes of almost all rats treated with 500 mg/kg after both 2 weeks and 4 weeks. Degenerative/necrotic germ cells and multinucleated giant cell formation were observed in 2 weeks, though to a lesser extent than in 4 weeks. On stage analysis of germinal cells in 2 weeks, spermatogonia and spermatids of stage VII were found to be decreased, and pachytene spermatocytes of stage X were increased. In conclusion, the results indicate that if the selection of doses is appropriate, testicular toxicity of boric acid can be detected even after only 2 weeks of repeated daily oral treatment.

Subacute toxicological study in monkeys treated orally with pravastatin sodium for 5 weeks.

Pravastatin sodium was administered orally to cynomolgus monkeys at dosage levels of 50, 100, 200 and 400 mg/kg/day for 5 successive weeks. Five animals of 200 mg/kg and 400 mg/kg dose groups were sacrificed during the study period, because these animals deteriorated in general condition and/or showed remarkable changes in serum biochemical examination. Pathological examination revealed hepatic and/or renal disturbance in these animals. These changes are thought to be the cause of deterioration of general condition or changes in serum biochemical examination. All other animals were terminated at the end of the study period. In the animals of the 100 mg/kg dose group, only one animal showed hepatic and renal disorder similar in nature to the changes in the animals sacrificed during the study period. No animals in the 50 mg/kg dose group showed toxic findings in any examination.

Heat curing behavior of light-cured composite resins investigated by dynamic differential scanning calorimetry.

The heat curing behavior of light-cured restorative composite resins, light-cured crown and bridge veneering resins, pure dimethacrylate monomers, Bis-GMA monomers containing various initiators, and monomer mixtures were investigated by slow heating at a constant rate with differential scanning calorimetry (DSC) without any light irradiation--the so-called dynamic DSC measurement. Some of the light-cured restorative composite resins and the light-cured crown and bridge veneering resins showed a sharp exothermic peak due to heat curing. Pure TEDMA also showed a sharp exothermic peak, whereas the Bis-GMA monomers containing catalysts for light curing showed no exothermic peak. It seems that heat curing behavior of light-cured composite resins depends not on the decomposition of camphorquinone, but on that of the monomer itself.

Genetic control of blood pressure in spontaneously hypertensive rats (SHR).

Genetic control of blood pressure in the SHR strain was studied by three separate experiments which consist of cross analysis between the SHR and Donryu, two-way selecton for high and low blood pressure levels, and successive backcrosses to the parental strains. The results obtained were as follows. 1. The data from genetic crosses between the SHR and Donryu showed the phenotype segregation ratio of 1:1 at the backcross and 1:2:1 at the F2 generation. 2. Two-way selection for high and low blood pressure levels was performed from the F2 generation onward. The separation between the two lines occurred immediately after the first selection. Thereafter, the difference increased gradually with generation. The blood pressure level at the seventh generation of selection became approximately equal to those of the parental strains. 3. Two types of the successive backcross were performed from the F1 hybrids by mating the males showing the highest blood pressure level to Donryu females and the females showing the lowest blood pressure level to SHR males on the other. Bimodality was observed in the distribution of blood pressure levels at each generation. Their phenotypic segregation ratios were accordant with 1:1 on the whole. At the intercross generation during successive backcrosses, a trimodal distribution was observed. 4. These results confirmed that the hypertensive trait of the SHR is regulated by a single major gene and other several genes with minor effect. A gene symbol ht was proposed for this major gene. Concurrently, a congenic strain having the ht gene on the genetic background of the Donryu was developed by the successive backcross system.

[Effect of proximate environment on drug susceptibility of mice (author's transl)].

The present study was performed in order to elucidate the effect of proximate environment on drug susceptibility of mice. Three experiments were carried out independently. In the first experiment, mongrel and ddS mice produced under an unsatisfactory control of proximate environment were purchased, and acute toxicity tests of thiamine hydrochloride (B1HCl) and isonicotinic acid hydrazide (INAH) were practiced at two different conditioned rooms. In the second experiment, ddY mice produced under the conventional environment controlled to a certain extent were purchased, and the toxic effect of B1HCl was examined under the similar environment. In the third experiment, the sensitivity to B1HCl of RFVL mice produced under the strict barrier system was tested at the severe air-conditioned room. LD50 and their fLD50 values were calculated by Litchfield-Wilcoxon's method, and the variance analyses were carried out. The severer the environmental control after the purchase of mice turned to the higher the drug sensitivity. This respect was more remarkable in INAH of which the toxic response is appeared slowly compared with B1HCl. Furthermore, seasonal variation was found in LD50 values. However, seasonal effect differed from rearing and experimental conditions. In the third experiment which these proximate environments were controlled severely, seasonal variation was very small. From the results of these experiments, it was defined that the use of animals produced under the satisfactory rearing condition and severe environmental control are necessary for animal experiments.

[4-way bred rats as experimental animals].

The present study is an attempt to utilize hybrids among several inbred strains of rats as useful animals for the studies of effectiveness and toxicology on drugs., Four-way crosses were made among the LEW, WM, F344 and DRY strains of rats, and their characteristics were examined. From the breeding data of diallel crosses among these four strains and reciprocal crosses among their F1 hybrids, the mating type indicating the highest reproductivity was (LEW X WM) F1 X (F344 X DRY) F1. These four-way crosses were designated as LWFD. The reproductivity of this mating type was exceedingly higher than those of four strains. In order to examine the susceptibility to thiamine hydrochloride, the acute toxicity test was practiced in inbred strains, F1 hybrids and four-way crosses. As a result, in spite of highly heterogeneous population, the LWFD did not show a peculiar response in comparison with four strains and their F1 hybrids. Furthermore, hematological and clinico-biochemical values of the LWFD did not show a large variability as presumed. From these results, it is suggested that hybrids such as four-way crosses among inbred strains can be used as useful animals for the studies of effectiveness and toxicology on drugs.

Studies on the genetic linkage of bilirubin and androsterone UDP-glucuronyltransferases by cross-breeding of two mutant rat strains.

Gunn rats, which have defects in bilirubin and 4-nitrophenol UDP-glucuronyltransferases (GT), were crossed with LA Wistar rats with a defect in androsterone GT. The F1 hybrids showed normal GT activities towards androsterone, bilirubin and 4-nitrophenol, demonstrating that Gunn and LA ('low activity') Wistar rats inherit a homozygous dominant trait for androsterone GT and bilirubin GT respectively. The F2 progeny showed four different combinations of bilirubin and androsterone GT activities: defects in both GT activities, a single defect in bilirubin GT activity, a single defect in androsterone GT activity and two normal GT activities. They were segregated in the approximate ratio of 1:3:3:9, which is compatible with Mendel's Principle of Independent Assortment. These results provide evidence that androsterone GT and bilirubin GT are located on different chromosomes. In the F2 generation, defective bilirubin and 4-nitrophenol GT activities were not segregated, indicating that these two mutant genes are closely linked on the same chromosome.

Augmented Ca2+ mobilization is a hypertensive trait discriminated from a 'major gene' in backcross analysis between SHR and Donryu rats.

1. Blood pressure and Ca2+ mobilization were significantly greater in SHR than in Donryu and F1 rats. 2. Backcross linkage analysis between spontaneously hypertensive rats and normotensive Donryu rats was performed to dissect polygenic hypertensive traits and to detect the existence of a single 'major gene'. 3. Cluster and discriminant analysis of a scattergram of blood pressure versus Ca2+ mobilization classified the backcrossed rats into two groups. The two groups were referred to the higher group and the lower group with regard to their relative blood pressure values. 4. Blood pressure was correlated with Ca2+ mobilization in each group; the correlation coefficients were 0.41 for the higher group (P < 0.01) and 0.71 for the lower group (P < 0.0001).

Genetic linkage of the sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase II gene to intracellular Ca2+ concentration in the spontaneously hypertensive rat.

A cosegregation analysis of sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (SERCA) II genotype, systolic blood pressure and platelet intracellular Ca2+ concentration was performed to dissect polygenic hypertensive traits in spontaneously hypertensive rats. Backcross analysis between spontaneously hypertensive rats and normotensive. Donryu rats demonstrated the existence of an inferred single major gene locus (ht). Thrombin-stimulated intraplatelet Ca2+ concentration was significantly higher in the SERCA II homozygotes than in the heterozygotes. The SERCA II genotype did not cosegregate with the blood pressure level. The SERCA II gene was assigned to rat chromosome 12. These results suggest that the SERCA II gene on rat chromosome 12 contributes to increased thrombin-stimulated intraplatelet Ca2+ concentration and that the SERCA II gene is not identical to ht.

A new neurological mutant rat with symmetrical calcification of Purkinje cells in cerebellum.

A new neurological mutant has been found in the inbred F344 strain of rats. The mutation is inherited as an autosomal recessive trait and is manifest clinically by a hesitant and wobbling gait with asynergic limbs and slight tremor. These symptoms begin at 16-18 days of age and remain essentially constant thereafter. Histologic examination revealed severe degeneration of the Purkinje cells and symmetrical calcification in these and in their dendritic branches in the cerebellar cortex. Such calcified Purkinje cells were intensely stained with the periodic acid-Schiff (PAS) method. PAS-positive substances in the Purkinje cells and extending diffusely over the lesioned sites in the molecular layer were also evident before calcification took place. We have named this neurological mutant the Cerebellar Calcification (CC) rat with the gene symbol cc. This offers a new animal model for the study of the Purkinje cell degeneration and intracranial calcification.

A comparison of uridine diphosphate-glucuronosyl-transferase and other drug metabolizing enzyme activities between two mutant strains of Wistar rats with a genetic deficiency in bilirubin or androsterone glucuronidation.

A jaundiced rat strain was derived from a cross between Gunn and Wistar-Imamichi rats, and inbreeding was continued by forced heterozygosis with jaundice locus. These Gunn rats have black pigment on heads and a black stripe on their backs similar to Long-Evans rats. Wistar rats with low activity in androsterone (AD) glucuronidation were selected and inbred (LA Wistar rats). The levels of hepatic uridine diphosphate-glucuronosyltransferase (GT) and sulfotransferase (ST) activities as well as cytochrome P-450 contents were compared in these mutant strains. Gunn rats were devoid of bilirubin (BL) GT activity but had high AD GT activity. LA Wistar rats had very low AD GT activity but showed high BL GT activity. Native and Triton X-100-stimulated GT activities toward 2-aminophenol and 4-nitrophenol (NP) were much lower in Gunn rats than in LA Wistar rats. When N-nitrosodiethylamine was added to the incubation media, these GT activities were stimulated equally to high levels in both mutants. N-Nitrodiethylamine provided a similar stimulatory effect on NP GT activity. There were no significant differences in ST activities toward cortisol, AD and NP and cytochrome P-450 contents in the two mutant strains. These results indicate that Gunn and LA Wistar rats have a different deficiency in GT isoenzymes.

Preclinical safety studies of cefmetazole.

In order to assess the safety of cefmetazole, preclinical multiple-dose parenteral studies, varying from one to three months in length, were conducted in Sprague-Dawley rats and beagle dogs. Although the largest doses used were in multiples of several times the weight-adjusted doses intended for humans, cefmetazole was generally well tolerated. The principal adverse effect noted in the adult rats receiving the largest doses (2000 and 2500 mg/kg/day) of cefmetazole was slight elevation of serum alanine aminotransferase. Infant rats injected subcutaneously with 300 mg/kg/day or more of cefmetazole for 35 consecutive days had reversible reductions in testicular weight and maturation of spermatogenesis, but not lasting discernible effect on reproductive function. The most consistent effects of longterm multiple dosing with cefmetazole in dogs consisted of vomiting and retching during dosing and reversible haematological changes (mild regenerative anaemia, positive Coombs' test, clinically-silent thrombocytopenia) in a number of the dogs. These findings supported the interpretation that cefmetazole was acceptably safe for clinical studies in humans.