To protect workers from the adverse effects of lead on haem synthesis and on the peripheral and central nervous system, a health-based biological exposure limit of 1.92 µmol/litre (40 µg/dl) was recommended by a WHO Study Group (WHO, 1980).
The steps within the haem biosynthetic pathway which have been used to measure effect are: 1) inhibition of delta-aminolaevulinic acid dehydratase (ALAD); 2) urinary excretion of delta-aminolaevulinic acid (ALAU); (3) the accumulation of zinc protoporphyrin (ZPP) in erythrocytes arising from the inhibition of the enzyme ferrochelatase or the iron transport system.
Mild dysfunction of the liver has been reported among Soviet workers engaged in the production of Na-PCP (Vinogradova et al., 1973) including, for example, a reduced ability to synthesize protein.
It has been suggested that delay in the accumulation of haemoproteins of the respiratory chain in brain tissue during development may result in decreased synthesis of haem enzymes in the brain (Bull, 1980; Moore et al., 1987).
Haem synthesis 220.127.116.11 Protoporphyrin levels 18.104.22.168 Coproporphyrin levels 22.214.171.124 delta-Aminolaevulinic acid levels in urine and blood 126.96.36.199 Aminolaevulinic acid dehydratase levels 188.8.131.52 delta-Aminolaevulinic acid synthase 184.108.40.206 Other effects of decreased haem synthesis
Small amounts of zinc 5X10-6 M stimulate rapid axonal transport of proteins in an in vitro system using frog ganglia and nerve (Edstrom and Mattsson, 1975). Zinc probably stimulates axonal transport by stabilizing rat brain microtubules and ribosomes (Edstrorn and Mattsson, 1975). Zinc, at concentration of 5X10-6 increased synthesis to 140 percent of the control and protein transport to 175 percent of the control value. In certain concentrations, Zn appears to be important for both protein synthesis and axoplasmic flow. Metal chelation of Zn causes nerve degeneration, while Zn toxicity causes fast axonal transport resulting in a distal concentration of membrane protein which may proceed to defective maintenance of axon terminal structures and loss of function.
To identify which one(s) of the mutations fixed in P25 and A25 populations caused the low fitness in BHK-21 cells we looked for changes present in A25 and P25 genotypes, but never found in regimes of adaptation to BHK-21 cells, including K25 genotypes. Within this group of mutations most or all are likely to be the result of selection, but some may be the result of hitch hiking. As indicated earlier, parallel mutations can be considered the product of selection because the likelihood of fixation in multiple replicas is extremely low. The probability of chance fixation for a neutral mutation is 1/Ne . Ne is the effective population size, and it can be calculated as the harmonic mean of the different population sizes during evolution . We can use the population size at transmission (the lowest value, 2 × 105 PFU) as Ne to calculate the maximum probability of fixation in a single replica of passages, so in our case the p value is 0.5 × 10-5. The probability of fixing the same neutral mutation in n independent replicas would be (0.5 × 10-5)n and thus we can ignore chance as a cause of parallel evolution. The presence of parallel mutations in the A25 and P25 genotypes, but not in BHK-21-adapted genotypes would represent antagonistic pleiotropy. In contrast, the presence of unique mutations can be interpreted in two different ways. First, they may be beneficial and also responsible for antagonistic pleiotropy. Second, they may be the result of hitch hiking, and cause tradeoffs due to mutation accumulation. Among the parallel mutations found in A25 and P25 strains, we found six mutations that never appeared in strains adapted to BHK-21 cells (). Substitutions A163G and G253A (N gene), C1817U (P gene) and C3978A (G gene) were non-synonymous. The N and P proteins have a role in RNA synthesis and mutations found in these proteins may have an impact in the observed phenotype. We also identified differences in the 3′ termini of the genomes: a 43-nt. duplication of nts. 20-62 at position 20, and a single substitution U→C at position 50 whose pattern of fixation was the same as that described for the A163G, G253A, C1817U and C3978A substitutions. Even though each of the four replicas of alternating regimes had a different combination of mutations, all the mutations in A25A, A25B and A25C were parallel (e.g. found in more than one replica) and likely the product of selection. This result ruled out any significant contribution of mutation accumulation to tradeoffs in these three genotypes. A25D had one unique mutation in the L gene (C8668U). On the basis of sequence comparison alone, this change could represent tradeoffs for BHK-21 infection in the form of mutation accumulation. However, there was no statistical difference between the fitness of A25D and any virus whose genotype lacked this individual mutations, that is, A25A, A25B and A25C (t test, p > 0.3). Thus, even if this unique mutation in A25D had no selective value, we could not assign any cost to it. In contrast, all the P25 genotypes had individual mutations, and their fitness in BHK-21 cells was significantly lower than the fitness of A25 genotypes. These individual mutations could be the result of selection or hitch hiking and, in the latter case, represent tradeoffs due to mutation accumulation. To test whether this was the case, we determined the sequence in persistent populations at passage 10 (P10).
Zinc deficiency has been shown to impair DNA, RNA and protein synthesis in the brains of suckling rats (Fosmire et al., 1975). Zinc deficiency results in impaired incorporation of thymidine into brain DNA. Incorporation of sulfur into protein is also decreased. Zinc deficiency also decreases the concentration of total lipid in brain while phospholipids and fatty acids are not affected.
Surprisingly, when sequencing the NSs gene, that encodes the virulence factor responsible for a general inhibition of cellular RNA synthesis and IFN-ß production, we found that single-host adapted viruses presented large deletions in this gene. Virus passaged in mammalian BHK21 cells defective in IFN-a/b signaling had a deletion of 259 nt introducing a stop-codon in the NSs gene. The resulting protein was shortened to 60 amino-acids instead of 265. Virus passaged in mosquito Aag2 cells showed a deletion of 73 nt, again with a stop-codon, causing a shortening of the NSs protein to 131 amino-acids. These two distinct deletions in the NSs gene following 30 serial passages in each cell type suggest that the viral genome may function differently depending on whether replication is in mammalian or mosquito cells. Thus, while serial passages of RVFV in a single cell type selected for a virus with a truncated NSs gene specific to that cell type, alternating passages did not allow the emergence of deletions in the NSs gene. Indeed, like the parental P strain, the 30th alternating passage virus Z30Alt did not present any major genetic changes in the NSs gene. The fact that the NSs gene is dispensable in both single host systems suggests that other mutations are involved in the host adaptation process. Thus, the different non-synonymous mutations identified in the segments M and L should be further explored in this context, particularly since they are mostly specialized depending on host. It is likely that these kinds of deletion events take place spontaneously during viral replication and are selected only in the absence of alternation. Further experiments involving independent serial passages would permit to evaluate the frequency at which the phenomenon occurs. Deletions in the NSs gene have been described in a naturally attenuated RVFV (Clone 13) purified from a nonfatal human case in the Central African Republic . This strain has a large internal deletion of 549 nt in the NSs gene (~70% of its length). Animals can survive high infectious doses of Clone 13, up to 106 PFU, without developing any symptoms. Furthermore, Clone 13 has been tested as a vaccine candidate in sheep and cattle that can indeed then elicit a protective response against challenge with a virulent RVFV strain. When the clones we obtained after 30 serial passages in a single cell type were inoculated into mice (at 104 PFU), the animals survived for 21 days and developed protective IgG against challenge with a virulent strain of RVFV. Moreover, most mice inoculated with Z30Alt (isolated at the 30th alternating passage) died 2 days later than did those treated with the parental P strain, and one mouse survived virus inoculation. Having said that, it is known that the outcome of infection is mainly determined by a balance between the rate of viral replication and the immune response, which together limit viral spread . This might explain why one of five mice recovered from infection. Nevertheless, the 30th alternating passage virus Z30Alt retained roughly the same level of virulence as the parental P strain owing to the maintenance of NSs integrity.