Rodent Mutation Research Articles

Automation: robots in the vivarium.

Automation technologies are improving efficiency in the vivarium and helping institutions keep up with the growing number of mutant rodents in their colonies.

Imaging sex/gender and autism in the brain: Etiological implications

The male preponderance in autism prevalence has brought together the disparate topics of sex/gender and autism research. Two directions of neuroimaging studies on the relationships between sex/gender and autism may inform male-specific risk mechanisms and female-specific protective mechanisms of autism. First, we review how sex/gender moderates autism-related brain changes and how this informs general models of autism etiology. Better-powered human neuroimaging studies suggest that the brain characteristics of autism are qualitatively, rather than simply quantitatively, different between males and females. However, age and comorbidities might substantially moderate the pattern of differences. Second, we review how the relationship between autism-related brain changes (separately in males and females) and normative brain sex/gender differences informs specific etiological-developmental mechanisms. Both human and animal studies converge to indicate that the brain characteristics of autism are partly associated with normative brain sex/gender differences, suggesting convergence or overlap between the mechanisms leading to and modifying the development of autism and the mechanisms underlying sex differentiation and/or gender socialization. Future animal work needs to investigate sex differences in rodent mutants modeling autism-relevant genes and environmental exposures. Future human work needs to address the substantial phenotypic and etiological heterogeneity of autism and to focus on longitudinal neuroimaging studies (from early development) on the developmental trajectories of sex/gender-differential neural characteristics of autism. Combining animal and human work links up the causal chain from etiological factors, brain and physical development, to phenotypes. These together help delineate the different roles of sex and gender in relation to risk vs. protective mechanisms. © 2016 Wiley Periodicals, Inc.

EFFECT OF GAMMA IRRADIATION ON PROTEIN PROFILE OF RODENT TUBER (TYPHONIUM FLAGELLIFORME LODD.) IN VITRO MUTANT BASED ON 1D AND 2D PAGE ANALYSES

Rodent tuber is an Indonesian anticancer herbal plant whose genetic variation is low. Genetic variation is crucial to be increased to increase the content of anticancer bioactive compounds. In vitro calli of rodent tuber had been mutated by gamma irradiation. Mutant plants had their genome modified and therefore also affected protein expression. The purpose of this research is to identify protein expression differences between mutant and control rodent tuber plants by 1D and 2D PAGE. 1D PAGE had shown differences in the number of protein bands between control and mutant. M6/2 did not show any expression of 6,9 kD protein. 2D PAGE had identified 24 proteins which were expressed by control and M6/2 plants. Among them, 13 spots were up-regulated and 9 spots were down-regulated in M6/2 plants. In conclusion, 1D and 2D PAGE analyses had successfully proven the changes in protein expression between control and mutant rodent tuber plants.

GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) ANALYSIS OF PHYTOCHEMICALS OF FIRST GENERATION GAMMA-IRRADIATED TYPHONIUM FLAGELLIFORME LODD. MUTANTS

Rodent tuber (Typhonium flagelliforme Lodd.) is an Indonesian herbal plant from Araceae family which is highly potential against several types of cancer. However, anticancer compound of rodent tuber is currently unknown. Rodent tuber has a low genetic diversity due to conventional vegetative propagation. In vitro propagation combined with gamma-irradiation of rodent tuber’s calli had been performed to increase the genetic diversity of rodent tuber. The mutant plants had been acclimatised and analysed with RAPD molecular markers, but the phytochemical constituents of these mutants have never been investigated. This research utilised gas chromatography-mass spectrometry (GC-MS) analytical method to identify and measure the relative abundances of major phytochemical constituents of rodent tuber control and mutant plants. GC-MS analysis successfully showed phytochemical constituents of the ethanol extract of rodent tuber plants. Shoots and tubers of mutant clones had at least 8 anticancer compounds whose quantities were higher than control plants. Shoots and tubers of mutant clones also contained new anticancer compounds which were not found in control plants. Shoots of mutant clones contained new anticancer compounds such as 7-pentadecyne, β-sitosterol, hexadecanoic acid methyl ester, cis-vaccenic acid, ergost-5-en-3-ol (campesterol). Tubers of mutant clones contained new anticancer compounds such as β-sitosterol, ethyl palmitate, hexadecanoic acid ethyl ester, vitamin E (alpha tocopherol), ergost-5-en-3-ol (campesterol). Rodent tuber mutant clones are therefore very potential to be developed as anticancer drugs.

Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia.

Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number of mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). MutaMouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia, and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, P < .01) and spermatogonial stem cells (2.1-fold at highest dose, P < .01). No significant increases in MF were detected in cauda sperm or seminiferous tubule cells collected 3 days post-exposure. Dose-response modelling suggested that the mutational response in male germ cells to BaP is sub-linear at low doses. Our results demonstrate that oral exposure to BaP causes spermatogonial stem cell mutations, that different phases of spermatogenesis exhibit varying sensitivities to BaP, with dividing spermatogonia representing a window of peak sensitivity, and that sampling spermatogenic cells from the seminiferous tubules at earlier time-points may underestimate germ cell mutagenicity. This information is critical to optimize the use of the international test guideline for transgenic rodent mutation assays for detecting germ cell mutagens.

The value of early and comprehensive diagnoses in a human fetus with hydrocephalus and progressive obliteration of the aqueduct of Sylvius: Case Report

BackgroundMutant rodent models have highlighted the importance of the ventricular ependymal cells and the subcommissural organ (a brain gland secreting glycoproteins into the cerebrospinal fluid) in the development of fetal onset hydrocephalus. Evidence indicates that communicating and non-communicating hydrocephalus can be two sequential phases of a single pathological phenomenon triggered by ependymal disruption and/or abnormal function of the subcommissural organ. We have hypothesized that a similar phenomenon may occur in human cases with fetal onset hydrocephalus.Case presentationWe report here on a case of human fetal communicating hydrocephalus with no central nervous system abnormalities other than stenosis of the aqueduct of Sylvius (SA) that became non-communicating hydrocephalus during the first postnatal week due to obliteration of the cerebral aqueduct. The case was followed closely by a team of basic and clinic investigators allowing an early diagnosis and prediction of the evolving pathophysiology. This information prompted neurosurgeons to perform a third ventriculostomy at postnatal day 14. The fetus was monitored by ultrasound, computerized axial tomography and magnetic resonance imaging (MRI). After birth, the follow up was by MRI, electroencephalography and neurological and neurocognitive assessments. Cerebrospinal fluid (CSF) collected at surgery showed abnormalities in the subcommissural organ proteins and the membrane proteins L1-neural cell adhesion molecule and aquaporin-4. The neurological and neurocognitive assessments at 3 and 6 years of age showed neurological impairments (epilepsy and cognitive deficits).Conclusions(1) In a hydrocephalic fetus, a stenosed SA can become obliterated at perinatal stages. (2) In the case reported, a close follow up of a communicating hydrocephalus detected in utero allowed a prompt postnatal surgery aiming to avoid as much brain damage as possible. (3) The clinical and pathological evolution of this patient supports the possibility that the progressive stenosis of the SA initiated during the embryonic period may have resulted from ependymal disruption of the cerebral aqueduct and dysfunction of the subcommissural organ. The analysis of subcommissural organ glycoproteins present in the CSF may be a valuable diagnostic tool for the pathogenesis of congenital hydrocephalus.

Evaluation of the sensitivity and specificity of in vivo erythrocyte micronucleus and transgenic rodent gene mutation tests to detect rodent carcinogens

Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e., Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames+in vivo MN (68.7%/45.3%), Ames+TGR (83.8%/not calculated (nc)), Ames+in vitro CA+in vivo MN (80.8%/21.3%), Ames+in vitro CA+TGR (89.1%/nc), Ames+in vivo MN+TGR (87.5%/nc), Ames+in vitro CA+in vivo MN+TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames+in vivo MN in comparison with Ames+in vitro CA (74.3%/37.5%). Ames+TGR and Ames+in vivo MN+TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens.

Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

BackgroundThe transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening.MethodsWe developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS.ResultsWe identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %.ConclusionThese results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2004-4) contains supplementary material, which is available to authorized users.

THE EFFECTS OF GAMMA IRRADIATION AND SOMACLONAL VARIATION ON MORPHOLOGY VARIATION OF MUTANT RODENT TUBER (Typhonium flagelliforme Lodd.) LINES

&lt;p&gt;Rodent tuber (Typhonium flagelliforme Lodd.) is an Indonesian plant with high medicinal potential as anti-cancer. This plant has a low genetic variation in Indonesia. Gamma irradiation can be used to increase genetic variation. This research aimed to explore the effects of gamma irradiation and somaclonal variation on several mutant rodent tuber lines. Six somatic cell populations, which were treated by 6 Gy of gamma irradiation, were successfully regenerated into plantlets. Six mutant lines had been sub cultured into second and third generations by using optimal regeneration media (MS0 supplemented with 0.5 mg/L BAP and 1 mg/L NAA). Rooting of in vitro plantlets had been done by using optimal rooting media MS0 supplemented with 0 mg/L NAA and 0.5 mg/L NAA). Plantlets with good roots were acclimatized and transferred into greenhouse. The morphology of first generation mutant in greenhouse (M1) were characterized and analyzed by using descriptive statistical method. The observed morphological characters including plant height, shoot number, leaf number, and leaf area. A hundred and seventy five (175) mutant lines were obtained on the third generation (MV3) from five somatic cell populations. Mutant line 6-3-x has the highest mean number of shoots, which was 4.62 shoots. Percentage of plant alive after acclimatization in the greenhouse was 78%. Out of 37 M1 mutant lines, morphologically diverse lines were observed with the highest plant height increase: &lt;br /&gt;15.5 cm on 6-2-4-1 mutant line, the highest plant shoots increase: 5 shoots on 6-6-7-8 mutant line, the highest leaf number increase: 17 leaves on 6-6-7-8 mutant line, and the highest leaf area increase: 47.24 cm2 on 6-2-5-2 mutant line. In conclusion, gamma irradiation and somaclonal variation could increase genetic variation of mutant rodent tuber lines as shown by morphological data. &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Keywords&lt;/strong&gt; : Thyphonium flagelliforme , Gamma-Irradiation Somaclonal-Variation, Morphology-Variation&lt;/p&gt;

MutAIT: an online genetic toxicology data portal and analysis tools.

Assessment of genetic toxicity and/or carcinogenic activity is an essential element of chemical screening programs employed to protect human health. Dose-response and gene mutation data are frequently analysed by industry, academia and governmental agencies for regulatory evaluations and decision making. Over the years, a number of efforts at different institutions have led to the creation and curation of databases to house genetic toxicology data, largely, with the aim of providing public access to facilitate research and regulatory assessments. This article provides a brief introduction to a new genetic toxicology portal called Mutation Analysis Informatics Tools (MutAIT) (www.mutait.org) that provides easy access to two of the largest genetic toxicology databases, the Mammalian Gene Mutation Database (MGMD) and TransgenicDB. TransgenicDB is a comprehensive collection of transgenic rodent mutation data initially compiled and collated by Health Canada. The updated MGMD contains approximately 50 000 individual mutation spectral records from the published literature. The portal not only gives access to an enormous quantity of genetic toxicology data, but also provides statistical tools for dose-response analysis and calculation of benchmark dose. Two important R packages for dose-response analysis are provided as web-distributed applications with user-friendly graphical interfaces. The 'drsmooth' package performs dose-response shape analysis and determines various points of departure (PoD) metrics and the 'PROAST' package provides algorithms for dose-response modelling. The MutAIT statistical tools, which are currently being enhanced, provide users with an efficient and comprehensive platform to conduct quantitative dose-response analyses and determine PoD values that can then be used to calculate human exposure limits or margins of exposure.

In vitro mammalian mutagenicity of complex polycyclic aromatic hydrocarbon mixtures in contaminated soils.

This study employed an in vitro version of the lacZ transgenic rodent mutation assay to assess the mutagenicity of nonpolar neutral and semipolar aromatic soil fractions from 10 PAH-contaminated sites, and evaluated the assumption of dose additivity that is routinely employed to calculate the risk posed by PAH mixtures. Significant mutagenic activity was detected in all nonpolar neutral fractions, and 8 of 10 semipolar aromatic fractions (nonpolar > semipolar). Mutagenic activity of synthetic PAH mixtures that mimic the PAH content of the soils (i.e., 5-PAH or 16-PAH mix) were greater than that of the PAH-containing soil fractions, with 5-PAH mix >16-PAH-mix. Predictions of mutagenic activity, calculated as the sum of the contributions from the mutagenic mixture components, were all within 2-fold of the observed activity of the nonpolar neutral fractions, with one exception. Observed differences in mutagenic activity are likely the result of dynamic metabolic processes, involving a complex interplay of AhR agonsim and saturation of metabolic machinery by competitive inhibition of mixture components. The presence of hitherto unidentified polar compounds present in PAH-contaminated soils may also contribute to overall hazard; however, these compounds are generally not included in current contaminated site risk assessment protocols.

Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea.

The transgenic rodent mutation assay was used to compare the dose–response relationship of lacZ mutant frequency (MF) in spermatogonial stem cells exposed acutely or subchronically to N‐ethyl‐N‐nitrosourea (ENU). Muta™Mouse males were exposed orally to 0, 25, 50, or 100 mg/kg ENU for acute exposures and 0, 1, 2, or 5 mg/(kg day) for 28‐day subchronic exposures. LacZ MF was measured in sperm collected 70 days post‐exposure to target spermatogonial stem cells. Dose–response data were fit to linear, quadratic, exponential, or power models. Acute exposure resulted in a dose‐dependent increase in MF that was significant (P < 0.05) at all doses tested and was best described by a quadratic dose–response model that was linear in the low dose range. In contrast, similar total doses fragmented over a 28‐day subchronic exposure only resulted in a significant increase in lacZ MF at the highest dose tested. Therefore, the subchronic no observable genotoxic effect level (NOGEL) was 2 mg/(kg day) (or 56 mg/kg total dose). The subchronic dose–response was best described by the exponential and power models, which were sublinear in the low dose range. Benchmark dose lower confidence limits (BMDLs) for acute and subchronic exposure were 3.0 and 1.0 mg/(kg day) (or 27.4 mg/kg total dose), respectively. These findings are supportive of a saturable DNA repair mechanism as the mutagenic mode of action for ENU in spermatogonia and imply that sufficiently low exposures would not cause appreciable genotoxic effects over background. This may have important implications for the quantitative risk assessment of germ cell mutagens. Environ. Mol. Mutagen. 56:347–355, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Evaluation of Rats' In Vivo Genotoxicity Induced by N-ethyl-N-nitrosourea in the RBC Pig-a, PIGRET, and gpt Assays

The emerging Pig-a gene mutation assay, a powerful and promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly those focused on peripheral RBCs and reticulocytes (RETs). Previously, it had been reported that Pig-a and gpt mutant frequencies were relatively increased in N-ethyl-N-nitrosourea (ENU)- and benzo[a]pyrene (BP)-treated mice. The capacity and characteristics of the Pig-a assay relative to transgenic rodent (TGR) mutation assays, however, are unclear in rats. Here, using transgenic gpt delta rats, we compared the in vivo genotoxicity of single oral doses of ENU (40 mg/kg) in the gpt gene mutation assay in bone marrow and liver, and Pig-a gene mutation assays on RBCs and RETs in the same animals. The Pig-a gene mutation assays were conducted at 1, 2, and 4 weeks after treatment, whereas gpt assays were conducted on tissues collected at the 4-week terminal sacrifice. Consequently, we detected that Pig-a and gpt mutant frequencies were clearly increased in ENU-treated rats, indicating that both the Pig-a and TGR gene mutation assays can detect in vivo ENU genotoxicity equally.

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea, benzo[a]pyrene, and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay.

New study design considerations for somatic and germ cell analysis in the Big Blue® transgenic rodent mutation assay

New study design considerations for somatic and germ cell analysis in the Big Blue® transgenic rodent mutation assay

MDR1-associated resistance to artesunate + mefloquine does not impair blood-stage parasite fitness in a rodent malaria model

If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin combination treatment (ACT) version of artesunate+mefloquine (ATN+MF) to gain insights about their ability to compete with ATN+MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN+MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN+MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.

D-Amino acids in the brain and mutant rodents lacking d-amino-acid oxidase activity

D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

Chromosome aberration frequency in rat peripheral lymphocytes increases with repeated dosing with hexamethylphosphoramide or cyclophosphamide

Although there are several in vivo tests for potential genotoxicity, with the possible exception of the transgenic rodent mutation models, none is specifically intended to assess increasing damage with chronic administration. In principle, peripheral blood lymphocytes would be expected to accumulate DNA damage with repeated dosing because the majority are not in active division and appear to have limited DNA repair capability, and they are exposed to plasma levels of test materials and metabolites. However, there appear to be no published reports confirming this principle. Therefore, in the current study, after optimising culture conditions for rat lymphocytes in this laboratory, rats were given oral doses of cyclophosphamide or hexamethylphosphoramide (HMPA) for up to 28 days and peripheral lymphocytes analysed for chromosome aberrations at various time points. The results clearly show that, for both compounds, doses that gave no significant increases in aberration frequency after 2 days induced clear increases after 15 days with further damage detectable after 28 doses. With HMPA, it was shown that DNA damage persisted for at least 10 days after cessation of treatment. These data show that repeat dose studies in the rat measuring chromosome aberration frequency in lymphocytes can give a genuine indication that genotoxicity may increase with chronic administration and, therefore, maybe useful in assessing the risk of potentially genotoxic substances.

Small-Scale Module of the Rat Granular Retrosplenial Cortex: An Example of the Minicolumn-Like Structure of the Cerebral Cortex

Structures associated with the small-scale module called “minicolumn” can be observed frequently in the cerebral cortex. However, the description of functional characteristics remains obscure. A significant confounding factor is the marked variability both in the definition of a minicolumn and in the diagnostic markers for identifying a minicolumn (see for review, Jones, 2000; DeFelipe et al., 2002; Rockland and Ichinohe, 2004). Within a minicolumn, cell columns are easily visualized by conventional Nissl staining. Dendritic bundles were first discovered with Golgi methods, but are more easily seen with microtubule-associated protein 2 immunohistochemistry. Myelinated axon bundles can be seen by Tau immunohistochemistry or myelin staining. Axon bundles of double bouquet cell can be seen by calbindin immunohistochemistry. The spatial interrelationship among these morphological elements is more complex than expected and is neither clear nor unanimously agreed upon. In this review, I would like to focus first on the minicolumnar structure found in layers 1 and 2 of the rat granular retrosplenial cortex. This modular structure was first discovered as a combination of prominent apical dendritic bundles from layer 2 pyramidal neurons and spatially matched thalamocortical patchy inputs (Wyss et al., 1990). Further examination showed more intricate components of this modular structure, which will be reviewed in this paper. Second, the postnatal development of this structure and potential molecular players for its formation will be reviewed. Thirdly, I will discuss how this modular organization is transformed in mutant rodents with a disorganized layer structure in the cerebral cortex (i.e., reeler mouse and shaking rat Kawasaki). Lastly, the potential significance of this type of module will be discussed.

Evaluation of the in vivo Mutagenicity of Nickel Subsulfide in the Lung of F344 gpt delta Transgenic Rats Exposed by Intratracheal Instillation: A Collaborative Study for the gpt delta Transgenic Rat Mutation Assay

This study was conducted to evaluate the effectiveness of a transgenic rat mutation assay using F344 gpt delta rats. We investigated the mutagenic potential in the lung of nickel subsulfide (Ni3S2), an insoluble fine-crystalline-metallic compound and a carcinogen in the rodent and human lung. Ni3S2 carcinogenicity has been proposed to act via both genotoxic and non-genotoxic mechanisms. Ni3S2 was intratracheally instilled into male gpt delta rats at doses of 0.5 and 1 mg/animal once a week for four weeks; these doses of Ni3S2 are high enough to induce inflammation in the lung. Following a period of 28 and 90 days after the first administration, the gpt mutant frequencies (MFs) in lung were determined in four independent laboratories, and Spi− selection for larger deletion mutations was done in one laboratory. The gpt MFs of the rats treated with Ni3S2 were not increased: all four laboratories obtained similar results with no statistical differences. The Spi− MFs were also not increased by exposure to Ni3S2. These results indicate that intratracheally instilled Ni3S2 is non-mutagenic in the lung of gpt delta transgenic rats; however, whether Ni3S2 is non-mutagenic in the lung or it induces mutations which are not detectable by transgenic rodent mutation assays requires further investigation.