Demetris Savva

Effects of dredging in Göteborg harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus).

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Göteborg harbor situated at the river Göta alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Göteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Göteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Göta alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.

Application of the polymerase chain reaction to the diagnosis of human toxoplasmosis

Toxoplasmosis may cause significant damage to the developing fetus and is a life-threatening opportunistic infection in immunocompromised persons. Serological investigation is unreliable, while isolation of the parasite is time consuming and may lack sensitivity. We have developed a system for detecting Toxoplasma gondii based on the amplification of the P30 gene using sequential rounds of PCR and nested primers. The clinical value of this technique was assessed by the investigation of a range of tissues taken from pregnant women, fetuses, neonates, AIDS patients and organ graft recipients. The PCR assay produced more positive reactions than isolation of the parasite by means of cell culture or animal inoculation. Extended autoradiography was found to be more sensitive than stained agarose gels for detecting the PCR product. Systematic contamination of PCR reactions was avoided but it was not possible to exclude sporadic contamination in certain cases. Detection of specific DNA is of clinical value in the investigation of the pregnant woman in order to assess the risk of transplacental passage of infection and in the fetus and neonate to identify congenital toxoplasmosis. Even so, PCR findings must be interpreted with caution because of the risk of a sample being contaminated. PCR may be the investigation of choice when brain biopsy is performed on a patient with AIDS and when toxoplasmosis associated with bone marrow transplantation is suspected.

Diagnosis of Cerebral Toxoplasmosis in Association with AIDS Using the Polymerase Chain Reaction

Two AIDS patients were provisionally diagnosed as having cerebral toxoplasma infection on the basis of compatible clinical presentation, serological evidence of exposure to toxoplasma and the detection of multiple space occupying lesions on CT scan. Initial response to conventional antitoxoplasma therapy was poor. Brain biopsy was performed and toxoplasma nucleic acid detected in the cerebral tissues utilising the polymerase chain reaction (PCR). Specific therapy was continued and a satisfactory clinical and radiological response was achieved in each case. The PCR represents a method of potential value for the diagnosis of cerebral toxoplasmosis associated with AIDS. Further studies are required to assess the sensitivity, specificity and prognostic value of this technique in comparison with established diagnostic methods.

Toxoplasma serology in Zambian and Ugandan patients infected with the human immunodeficiency virus

In the USA and Europe, toxoplasmosis is well recognized as an important cause of morbidity and mortality among immunocompromised individuals. Toxoplasma gondii has been shown to be a common opportunistic infection in patients infected with the human immunodeficiency virus (HIV) in the USA and Europe with published estimates ranging from 20% to 80%. The importance of Toxoplasma infection in East Africa has not yet been defined. The seroprevalence rates of toxoplasmosis in Zambian and Ugandan patients were determined using the dye test (DT) and the latex agglutination test (LAT). The geographical variation in seroprevalence rates noted in western countries was also found in these African countries, with Zambia showing significantly lower rates than Uganda. 34% of Ugandan (64/186) and 4% of Zambian (8/187) patients infected with HIV, compared with 27% of Ugandan (26/93) and 11% of Zambian (20/189) HIV-negative persons, had anti-Toxoplasma immunoglobulin G antibodies. With the LAT, 13% of the Ugandan and 7% of the Zambian sera gave a false positive result. The relevance of Toxoplasma serology in Africa is discussed.

The Use of Arbitrarily Primed PCR (AP-PCR) Fingerprinting to Detect Exposure to Genotoxic Chemicals

Exposure of an organism to a genotoxic chemical may result in the formation of covalently bound adducts between the chemical (or its metabolites) and the DNA; faulty repair of these adducts often results in mutations and, sometimes, cytogenetic changes. The primary effects of such exposure (i.e. adduct formation) and the subsequent effects on the DNA (mutation, cytogenetic damage) may be monitored using a number of assays of varying sensitivity and specificity. Recent developments in molecular biology offer new possibilities for detecting DNA damage. In this laboratory DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) was investigated in order to establish whether it can reveal differences in the DNA fingerprints of animals exposed to benzo[a]pyrene in the laboratory and of animals from control and from polluted areas. The results indicate that differences between control and exposed animals were detectable; these results, together with those from other laboratories, indicate that DNA fingerprinting by AP-PCR offers a useful alternative biomarker assay for the detection of the genotoxic effects of environmental pollutants. This paper reviews the application of PCR based DNA fingerprinting procedures in mutation detection and discusses their application to ecotoxicological studies.

Expression and partial purification of human prolactin in escherichia coli

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.

Characterisation of Two Metallothionein cDNAs from the Shore Crab for use as Biomarkers of Heavy Metal Pollution

Molecular biological procedures open up possibilities for the development of new biomarker assays for use in environmental monitoring studies. Metallothionein (MT) is a useful biomarker for monitoring pollution by heavy metals and since very little information is available on the genes for MT in marine invertebrates, studies have been initiated in order to develop probes for use in biomarker assays for MT in the shore crab (Carcinus maenas). RNA isolated from the gills of shore crabs was used to produce complementary DNA (cDNA) from which two incomplete and two complete MT cDNAs have been isolated and characterised. The first complete cDNA (cDNA-4) encodes for a protein of 58 amino acids with a predicted molecular mass of 6151 Da; the predicted amino acid sequence of this protein is identical to that determined earlier for MT-Ib isolated from cadmium-exposed crabs. The second complete cDNA (cDNA-3) encodes a protein of 41 amino acids with a predicted molecular mass of 4484 Da; only the 5 C-terminal residues of this truncated MT differ from those at the corresponding positions of MT-Ib and this may correspond to a 4100 Da MT also reported previously. The implications of these findings and the use of these cDNAs as biomarkers in ecotoxicological studies are discussed.

Detection of foreign DNA in transgenic chicken embryos using the polymerase chain reaction.

Chicken primordial germ cells were infected with a defective retrovirus containing the Escherichia coli lacZ gene and injected into the heart of stage 15 embryos. DNA samples were isolated from various tissues of the injected embryos at different stages of development and were examined for the presence of the lacZ gene using the polymerase chain reaction. Integration of the retrovirus DNA was demonstrated with a 32P-labelled oligonucleotide in five-, 10- and 18-day embryos. This quick procedure provides an opportunity for the early detection of foreign DNA in small numbers of transfected cells and is a valuable tool in the detection of transgenic animals.

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: Characterization and purification

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Accumulation of Mutagenic Xenobiotics in Fresh Water (Lake Baikal) and Marine (Hornoya Island) Ecosystems

Extracts from tissues of a wide range of aquatic organisms (plants, plankton, decapods, molluscs, fish, Baikal seals, and fish-eating birds and their eggs) from Lake Baikal and from the Selenga River estuary and tissue extracts of birds breeding on Hornoya Island (northern Norway) were assayed for mutagenicity using the Ames Salmonella/microsome test. The activities of cytochrome P-450 and the enzymes of phase II of detoxification were also studied in the liver of fish and birds. Evidence was found for the accumulation of mutagens in the food chain. The relationship of bioaccumulation to the levels of enzyme activities possessing both detoxification and activation functions is discussed in the cases of fish and birds. The accumulation of mutagens was found to depend on the activity and the level of induction of the enzymes providing the detoxification and metabolic activation in the livers of fish and birds.