Toxicity testing in the 21st century: progress in the past decade and future perspectives.

Advances in the biological sciences have led to an ongoing paradigm shift in toxicity testing based on expanded application of high-throughput in vitro screening and in silico methods to assess potential health risks of environmental agents. This review examines progress on the vision for toxicity testing elaborated by the US National Research Council (NRC) during the decade that has passed since the 2007 NRC report on Toxicity Testing in the 21st Century (TT21C). Concomitant advances in exposure assessment, including computational approaches and high-throughput exposomics, are also documented. A vision for the next generation of risk science, incorporating risk assessment methodologies suitable for the analysis of new toxicological and exposure data, resulting in human exposure guidelines is described. Case study prototypes indicating how these new approaches to toxicity testing, exposure measurement, and risk assessment are beginning to be applied in practice are presented. Overall, progress on the 20-year transition plan laid out by the US NRC in 2007 has been substantial. Importantly, government agencies within the United States and internationally are beginning to incorporate the new approach methodologies envisaged in the original TT21C vision into regulatory practice. Future perspectives on the continued evolution of toxicity testing to strengthen regulatory risk assessment are provided.

The antifreeze potential of the spruce budworm thermal hysteresis protein.

Antifreeze proteins (AFP) inhibit ice growth by surface adsorption that results in a depression of the freezing point below the melting point. The maximum level of this thermal hysteresis shown by the four structurally unrelated fish AFP is approximately 1.5 degrees C. In contrast, hemolymph and crude extracts from insects can have 5 degrees to 10 degrees C of thermal hysteresis. Based on the isolation, cloning, and expression of a thermal hysteresis protein (THP) from spruce budworm (Choristoneura fumiferana), the vastly greater activity is attributable to a 9 kDa protein. This novel, threonine- and cysteine-rich THP has striking effects on ice crystal morphology, both before and during freezing. It is also 10 to 30 times more active than any known fish AFP, offering the prospect of superior antifreeze properties in cryoprotective applications.

Bovine spongiform encephalopathy and the safety of milk from Canadian dairy cattle.

The detection of bovine spongiform encephalopathy (BSE) in beef cattle closed Canadian beef export markets to 30 countries, including the USA, with devastating financial losses. The detection and confirmation of the fifth and seventh BSE-infected animals but first infected dairy cows extended the problem of risk management to Canadian dairy farmers. As the public are concerned about the safety not only of beef but also of milk and milk products that may contain disease-causing prions, this review examines the evidence for the safety of milk from studies on prions in milk or colostrum and their vertical and lateral transmission in various animal systems. The evidence indicates that the risk of contracting new variant Creutzfeldt-Jakob disease through the consumption of milk is negligible.

Towards a reconciliation of the introns early or late views: triosephosphate isomerase genes from insects.

The gene encoding the glycolytic enzyme, triosephosphate isomerase (TPI; EC 5.3.1.1), is a favourite model for molecular evolutionists who either subscribe to the theory that introns co-evolved with the ancestral gene, the introns early view, or alternatively, that introns are more recent immigrants. The discovery of an intron in the TPI gene of Culex mosquitoes at a site which was predicted by proponents of the intron early school supported that theory. More recently, the discovery of additional intron sites in several eukaryotes was presented as evidence supporting the introns late school. We have found the 'Culex intron' in two closely related mosquitoes, but not in two more evolutionary primitive Dipterans, suggesting that, if it is an 'ancient intron', loss may be more frequent than that supposed by the intron late school. In addition, we have found that three introns punctuating the TPI gene from the Lepidopteran, Heliothis, appear to be ancestrally related and may be the result of transposable element insertion, 50-90 million years ago. It is argued that both opposing schools in the intron debate be reconciled -- some introns may have been early and certainly others have arrived subsequent to the appearance of the TPI gene.

Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells.

Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line. Uptake experiments with [3H]MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line. Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line. To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells. Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells. Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme. Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX. These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects.

Transcription of antifreeze protein genes in Choristoneura fumiferana.

Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.

Analysis of antifreeze proteins within spruce budworm sister species.

Spruce budworm (Choristoneura) species survive sub-zero winter temperatures by producing antifreeze proteins (AFPs) encoded by a multigene family of short and long isoforms. We report in this study the first analysis of antifreeze proteins from related Choristoneura sister species. The additional thirty amino acid insert found in the longer AFP isoforms maintains the proteins beta-helix and original fifteen amino acid (Thr-X-Thr) repeat motif. Analysis of the beta-helix region shows more divergent residues surround the conserved Thr residues. Maintaining the beta-helix structure and conserved Thr residues appear to be paramount for AFP function and surviving sub-zero winter temperatures. Two other species within the same lepidopteran clade, Ditrysia, do not appear to contain any AFP-like sequences.

Dihydrofolate reductase of Drosophila. Cloning and expression of a gene with a rare transcript.

Traditionally, dihydrofolate reductase (DHFR) has been isolated and the corresponding gene cloned from drug-resistant cell lines which have amplified DHFR genes after selection. A Dhfr sequence has now been obtained by nested polymerase chain reaction (PCR) from Drosophila bearing a single gene copy. Using the PCR-amplified partial cDNA as a probe, Dhfr was cloned by screening a Drosophila genomic library. It consists of regulatory regions as well as a 599-nucleotide coding region with a single 50-base pair (bp) intron and encodes a protein of 182 amino acids. Previously we have shown that the enzyme has kinetic properties characteristic of both "prokaryotic" and "eukaryotic" DHFRs. Here we show that the organization of Drosophila Dhfr is strikingly different from its mammalian counterparts and most similar to that of mosquito. A 790-bp transcript was detected by Northern blot analysis, with a single transcription start site located 27 bp upstream of ATG codon. The Drosophila genome contains a single Dhfr copy at 89E and a selected cell line has not amplified the gene. Confirmation of the identity of this gene has been obtained by kinetic studies of recombinant DHFR over-expressed in Escherichia coli cells.

Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes. Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes--the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens--and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations. The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.

A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line.

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were approximately 1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing approximately 400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

Tobacco budworm dihydrofolate reductase is a promising target for insecticide discovery.

Structural differences in dihydrofolate reductases from different species have been exploited to develop specific inhibitory molecules, such as chemotherapeutic agents, antibiotics or antihelminthics, that show species specificity or selectivity. As dihydrofolate reductase (DHFR) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also because developing insects show a remarkably rapid rate of cell division, DHFR is a potentially promising target for the discovery of novel insecticides. We have thus isolated and characterized the enzyme from a serious agricultural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequencing tryptic peptides of the 35 000-fold purified DHFR allowed the subsequent isolation of a partial cDNA, with the full Dhfr gene sequence obtained from a genomic library. The H. virescens Dhfr spans 4 kb, with three introns, and encodes 185 amino acids. The enzyme shows an overall similarity of approximately 68% with DHFR from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs from insects appear to have strikingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total binding energy seen after modeling experiments. Four residues that may be characteristic of insect DHFR, as well as a unique cysteine in the H. virescens DHFR active site, offer insight into the nature of inhibitor selectivity and provide suitable target sites for insecticide discovery.