H. van Steeg

Targeting efficiency of a mitochondrial pre-sequence is dependent on the passenger protein.

The mitochondrial matrix enzyme manganese superoxide dismutase (SOD) of Saccharomyces cerevisiae is encoded in the nucleus. It is synthesized as a precursor with an NH2‐terminal extension of 26 amino acids which is cleaved off during import into the mitochondrion. Fusions between the NH2‐terminal 34 amino acids of SOD and the cytosolic proteins invertase of yeast and mouse dihydrofolate reductase (DHFR) were tested for in vitro binding and import into mitochondria. Efficient translocation over the mitochondrial membranes takes place in the case of the SOD‐DHFR fusion. The SOD‐invertase fusion protein does not get translocated and binds to the organelle with only low efficiency. Yeast transformants harbouring the SOD‐invertase fusion gene accumulate approximately 95% of the hybrid protein in the cytosol. The remaining material is found in the interior of the mitochondrion, loosely attached to the inner membrane. We conclude that the pre‐sequence of SOD is able to deliver a passenger protein to the mitochondrion. The efficiency of protein delivery and translocation across the membrane is, however, influenced by the passenger protein.

RNA editing in transcripts of the mitochondrial genes of the insect trypanosome Crithidia fasciculata.

With the aid of cDNA and RNA sequence analysis, we have determined to what extent transcripts of mitochondrial maxicircle genes of the insect trypanosome Crithidia fasciculata are altered by RNA editing, a novel mechanism of gene expression which operates via the insertion and deletion of uridine residues. Editing of cytochrome c oxidase (cox) subunit II and III transcripts and of maxicircle unidentified reading frame (MURF) 2 RNA is limited to a small section and results in the creation of a potential AUG translational initiation codon (coxIII, MURF2) or the removal of a frameshift (coxII). No differences with the genomic sequences were observed in the remainder of these RNAs. Surprisingly, NADH dehydrogenase subunit I transcripts were completely unedited in the coding region, implying that an AUG translational initiation codon is absent. The partial ribosomal RNA sequences determined also conform to the gene sequences. Together these results lead to the conclusion that the unusual sequences predicted by the protein and rRNA genes must indeed be present in the gene products. Editing also occurred in the poly(A) tail of RNAs from all protein genes, including those that are unedited in the coding region. The tails display a large variation in AU sequence motifs. Finally, some cDNAs contained sequences absent from both the DNA and the edited RNA. Some of these may represent intermediates in the RNA editing process. We argue, however, that long runs of T may be artefacts of cDNA synthesis.

Gene Expression Profiling of Bis(tri-n-butyltin)oxide (TBTO)-Induced Immunotoxicity in Mice and Rats

Bis(tri-n-butyltin)oxide (TBTO) is one of the organotin compounds that have been used as biocides and occur as persistent environmental pollutants. Human exposure to these compounds occurs through consumption of meat and fish products in which they accumulate. The most sensitive endpoint of TBTO exposure is immunotoxicity. TBTO causes thymus atrophy and thereby interferes with T-lymphocyte-mediated immune responses. Tributyltin compounds have been found to adversely affect a wide range of cellular components and processes in many species, organ systems, and cell types. Both inhibition of proliferation and induction of apoptosis have been observed in thymocytes. We conducted microarray experiments in mice and rats in order to investigate if the immunosuppressive actions of TBTO could be detected by gene expression profiling, and if so, to elucidate the mechanisms of action. Gene expression changes that were detected in mouse thymuses after exposure to a maximum tolerable dose of TBTO correlated to previously observed effects. Most notably, reduction of expression of cell surface determinants and T-cell receptor chains, suppression of cell proliferation, and a possible involvement of nuclear receptors in interference with lipid metabolism by TBTO were observed. The TBTO-induced thymus involution may therefore primarily be caused by inhibition of thymocyte proliferation. In contrast, in rats only limited effects of a lower dose of TBTO were found at the gene expression level in the thymus, even though thymus involution was observed. Here, most gene expression regulation by TBTO was detected in the liver. These preliminary results indicate that gene expression analysis is able to reveal effects of TBTO and to gain insight into its molecular mechanism of action. It may even be a suitable tool to investigate immunotoxicology in general. However, dose and inter-species differences are apparently clearly reflected in the gene expression profiles.

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8‐fold difference between the molar ratio of these two gRNAs relative to the (pre)‐mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5′ end but with a different 3′ uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing‐like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3′‐terminus to an editing site of pre‐edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5′ end of pre‐edited RNA.

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: Toxicokinetics is essential for in vivo–in vitro comparisons

The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa−/−p53+/− primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa−/−p53+/− hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa−/−p53+/− mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa−/−p53+/− livers only. In order to be able to make a meaningful in vivo–in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa−/−p53+/− exposed livers, which were previously found to be induced by B[a]P in Xpa−/−p53+/− primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.

Genotoxic exposure: novel cause of selection for a functional ΔN-p53 isoform

The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogenesis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform ΔN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of ΔN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the ΔN-p53 isoform in a p53-null background are divergent from p53 knockout ES cells, and therefore postulate that ΔN-p53 itself has functional transcriptional properties.

Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis

The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time‐consuming and animal‐demanding process. Introducing microarray‐based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene‐expression responses with a recognizable overlap, these gene‐expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non‐reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene‐expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non‐reprotoxic counterparts. This shows that differences in gene‐expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for ‘early warning’ biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost‐effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation. Copyright

Unraveling Stochastic Aging Processes in Mouse Liver: Dissecting Biological from Chronological Age

Abstract Cellular damage accumulation is a central feature of aging, resulting in functional decline and increased vulnerability to pathology and disease. This accumulation occurs over time, but is not exclusively time-dependent. Recent studies showed that, concerning biomedical issues, age may rather be defined as biological age, expressed as the cumulative change of physiological or functional parameters, in contrast to chronological age. We describe a method that goes beyond chronological age as the primary determinant for aging in the investigations of mechanisms underlying aging. We show in mice that the use of biological phenotypes reveals processes, genes, and pathways related to aging that would not have been revealed when using chronological age as the main determinant of aging. Using determinants of biological aging will improve our knowledge regarding the mechanisms underlying aging and may lead to the discovery of new biomarkers of frailty valuable for predicting health risks.