Wagida A. Anwar

The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt

BACKGROUND The population of Egypt has a heavy burden of liver disease, mostly due to chronic infection with hepatitis C virus (HCV). Overall prevalence of antibody to HCV in the general population is around 15-20%. The risk factor for HCV transmission that specifically sets Egypt apart from other countries is a personal history of parenteral antischistosomal therapy (PAT). A review of the Egyptian PAT mass-treatment campaigns, discontinued only in the 1980s, show a very high potential for transmission of blood-borne pathogens. We examine the relative importance of PAT in the spread of HCV in Egypt. METHODS The degree of exposure to PAT by cohort was estimated from 1961-86 Ministry of Health data. A cohort-specific exposure index for PAT was calculated and compared with cohort-specific HCV prevalence rates in four regions. FINDINGS HCV prevalence was calculated for 8499 Egyptians aged 10-50 years. A significant association between seroprevalence of antibodies to HCV and the exposure index (1.31 [95% CI 1.08-1.59]; p=0.007) was identified across four different regions. In all regions cohort-specific HCV prevalence was lowest in children and young adults than in older cohorts. These lower prevalence rates coincided with the gradual and final replacement of PAT with oral antischistosomal drugs at different points in time in the four regions. INTERPRETATION The data suggest that PAT had a major role in the spread of HCV throughout Egypt. This intensive transmission established a large reservoir of chronic HCV infection, responsible for the high prevalence of HCV infection and current high rates of transmission. Egypts mass campaigns of PAT may represent the worlds largest iatrogenic transmission of blood-borne pathogens.

A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies

A deletion polymorphism in glutathione S-transferase theta (GSTT1) gene was recently discovered in humans. Similar to the GSTM1 gene, GSTT1 is also recognized as a risk modifier in exposed populations. To evaluate the role of genetic polymorphism in health effects, the combined genetic polymorphism of different genes should be taken into consideration. In the present study, we have developed a multiplex PCR approach for simultaneous replication of both genes for molecular analysis. The multiplex PCR protocol was validated using donor DNA with different polymorphic combinations for both genes from two different ethnic populations (North Americans and Egyptians). The prevalence of the GSTM1 null genotype was 51% among North Americans and 44% among Egyptians. The prevalence of the GSTT1 null genotype was 15% among North Americans and 14.7% among Egyptians. Combined polymorphism analysis of both genes revealed that 6.3% of North Americans harbor the deleted genotype of both genes compared to 8.8% of the Egyptians. The data indicate that there is no major difference in allelic distribution of both genes between the ethnic populations. The multiplex PCR assay used in this study has the advantage of reducing the time, effort and cost required to carry out such analysis. It will also significantly enhance the ability to use genetic screening techniques as a potential tool for early detection of health outcomes in exposed populations.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

Involvement of inflammatory reactions and elevated cell proliferation in the development of bladder cancer in schistosomiasis patients

Schistosoma haematobium infection is strongly associated with urinary bladder cancer. Although numerous explanations have been proposed for this association, the nature of this relationship remains unresolved. This paper explores the hypothesis that inflammation and elevated cell proliferation play a major role in the development of bladder cancer in infected patients, possibly by increasing the level of genetic instability in the urothelium. The paper details in vivo and in vitro studies being done in our laboratories to test this hypothesis. These studies include population studies in which chromosomal breakage in the bladder of infected individuals is assayed using the micronucleus (MN) test on exfoliated urothelial cells. The approach also includes parallel studies in Vancouver with patients with long-term catheter drainage, a population with many similarities to schistosomiasis patients. In the in vitro studies we are co-incubating bladder cells with activated neutrophils or experimental conditions simulating inflammation. These studies show that inflammatory cells when activated can induce micronuclei in bladder cells and that this response is associated with loci on chromosome 11, a chromosome commonly altered during bladder carcinogenesis. A final approach being used is to assay chromosomal change (MN frequencies and numerical chromosome alterations) and level of proliferation (expression of proliferating cell nuclear antigen) in archival biopsies from schistosomiasis patients. Preliminary results show that a dysregulation of cell proliferation is occurring during cystitis in these patients. The extent to which this alteration affects the level of chromosomal breakage is yet to be determined.

Changing pattern of hepatocellular carcinoma (HCC) and its risk factors in Egypt: Possibilities for prevention

The burden of hepatocellular carcinoma (HCC) has been increasing in Egypt with a doubling in the incidence rate in the past 10 years. This has been attributed to several biological (e.g. hepatitis B and C virus infection) and environmental factors (e.g. aflatoxin, AF). Other factors such as cigarette smoking, occupational exposure to chemicals such as pesticides, and endemic infections in the community, such as schistosomiasis, may have additional roles in the etiology or progression of the disease. Estimates of the burden of cancer caused by these factors provide an opportunity for prevention. Previously, there was strong evidence that hepatitis B virus (HBV) was the major cause of HCC in Egypt, but more recently HCV has become the predominant factor associated with the more recent epidemic of HCC. It has been well documented that Egypt has one of the highest prevalence rates of HCV infection in the world. The natural history of HCV infection and disease progression, however, are influenced by additional factors such as duration of infection, age at infection, sex, co-infection with HBV, the level of HCV viraemia and its genotype. The role of exposure to aflatoxins and development of HCC in Egypt was historically less clear. Nevertheless, recent food sampling surveys and population-based studies indicated that exposure to aflatoxins in Egypt may have been underestimated in the past. Recent results indicated that both local and imported samples were positive for aflatoxin B1 (AFB1, 17.5% and 20%, respectively), with concentrations ranging from 3 to 25 microg/kg. The level of AFB1 was dependent on the area of collection as well as the season of the year. In a population-based study, the level and frequency of aflatoxin M1 (AFM1, a major metabolite of aflatoxin B1 excreted in breast milk) was assessed as a biomarker of maternal exposure. The samples were collected from a selected group of 388 Egyptian lactating mothers during May-September 2003. Non-working status, obesity, high corn oil consumption, and the number of offspring contributed to the variability in occurrence of AFM1 in breast milk. Prevention and intervention approaches directed to risk factors of HCC can play a critical role in its prevention. In the case of HCV infection a prevention programme can be achieved by changing personal behaviors and/or cultural habits which are risk factors for HCV transmission, such as injection with contaminated syringes, blood transfusion, surgical operations, venous catheterization, use of common syringes, dental treatment and circumcision at home. Prevention of exposure to aflatoxins can be achieved either at community (via good agriculture practices) or individual levels (treatment or dietary interventions). In conclusion, due to the alarming increase in the incidence of HCC in Egypt, there is a need to further investigate the contribution of these emerging risk factors to the development of HCC in Egypt. This may enable us to determine the susceptibility to HCC among high-risk groups and to provide these individuals with effective measures for early prevention or intervention.

The mutagenicity of Gramoxone (paraquat) on different eukaryotic systems

The possible mutagenicity of the herbicide Gramoxone was evaluated using five different living systems: Allium cepa, Vicia faba, yeast, Drosophila melanogaster and human lymphocytes. The results indicate that Gramoxone has mutagenic activity at the cytological level in Allium cepa, Vicia faba and human lymphocytes. All doses were effective in inducing chromosomal abnormalities and a clear dose-response relationship was observed in the various cytological tests. Analysis of chromosomal abnormalities revealed that this herbicide displays clastogenic and turbagenic activities. At the gene mutation level Gramoxone induced gene conversion at the trp-5 locus and reversion at the ilv locus in Saccharomyces cerevisiae. In Drosophila melanogaster, Gramoxone proved to be mutagenic to germ cells and induced a high frequency of sex-linked recessive lethals (SLRL). At the protein level, Gramoxone had detectable mutagenic effects on the genetic background of two enzymes, Adh and Est-6. Gramoxone should be considered a mutagenic herbicide.

Environmental health in Egypt

Egypt shares most of the environmental problems of developing countries. One of the most important health and environmental problems is air pollution resulting from using fuel, burning operations, and the increase of automobile exhaust in cities. Moreover, the deficiency of efficient sanitation services and water pollution caused by the breaking down of old and consumed water networks, as well as the various problems in construction, designing and maintenance of sewage system resulted in the appearance and prevalence of communicable and non-communicable diseases. There are several examples of exposure to chemical genotoxicants, and lifestyle exposures in the population, which create unique combinations of environmental risk factors for diseases such as cancer. Environmental factors may interact with infection and lead to enhancement of carcinogenicity processes. Currently, there is a growing interest in environmental mutagenicity and carcinogenicity research. The use of different biomarkers and genetic susceptibility testing can contribute effectively to risk assessment. The Government of Egypt recognizes and deals seriously with these problems. The State Ministry of Environment has initiated new policies that include risk minimization, law enforcement, treatment of pollution at source, mitigation procedures and inter-sectorial collaboration. The Ministry of Health and Population recognized the link between economic development, environment and health. It elaborated a national environment health strategy in accordance with the format of the regional and global environmental health policy. This strategy identified priority areas, which requires further action to be taken and to be implemented. Environmental health was included as one of the four main objectives of the strategic Healthy Egyptians 2010 Initiative. Specific objectives and plans for the initiative are presented.

HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems

The use of micronucleus (MN) assays in in vitro genetic toxicology testing, radiation biodosimetry and population biomonitoring to study the genotoxic impacts of environment gene-interactions has steadily increased over the past two decades. As a consequence there has been a strong interest in developing automated systems to score micronuclei, a biomarker of chromosome breakage or loss, in mammalian and human cells. This paper summarises the outcomes of a workshop on this topic, organised by the HUMN project, at the 6th International Conference on Environmental Mutagenesis in Human Populations at Doha, Qatar, 2012. The aim of this paper is to summarise the outcomes of the workshop with respect to the set objectives which were: (i) Review current developments in automation of micronucleus assays by image cytometry; (ii) define the performance characteristics of automated MN scoring using image cytometry and methods of assessment for instrument validation and quality control and (iii) discuss the design of inter-laboratory comparisons and standardisation of micronucleus assays using automated image cytometry systems. It is evident that automated scoring of micronuclei by automated image cytometry using different commercially available platforms [e.g. Metafer (MetaSystems), Pathfinder™ (IMSTAR), iCyte(®) (Compucyte)], particularly for lymphocytes, is at a mature stage of development with good agreement between visual and automated scoring across systems (correlation factors ranging from 0.58 to 0.99). However, a standardised system of validation and calibration is required to enable more reliable comparison of data across laboratories and across platforms. This review identifies recent progress, important limitations and steps that need to be taken into account to enable the successful universal implementation of automated micronucleus assays by image cytometry.

Micronuclei, chromosomal aberrations and aflatoxin-albumin adducts in experimental animals after exposure to aflatoxin B1

Rats and mice differ markedly in sensitivity to aflatoxin B1 (AFB1) hepatocarcinogenicity, the former being sensitive and the latter resistant. Animals were treated with single doses of different concentrations of AFB1, between 0.01 and 1.0 microgram AFB1/g body weight. The frequency of chromosomal aberrations and micronuclei in the bone marrow was measured and compared to the level of AFB1 bound covalently to albumin in the peripheral blood. Both chromosomal aberrations and micronuclei were significantly increased in treated rats compared to the control group at doses above 0.1 microgram/g. In contrast, in mice, a slight increase in chromosome aberrations was seen in the highest dose group (1.0 microgram/g) but no increase in micronuclei was observed at any of the doses. The level of chromosomal aberrations was about 10 times higher in rats than in mice at the highest dose of AFB1. AFB1-albumin (AF-alb) adducts did not show a strong dose-response increase after treatment in mice, whereas in rats the levels increased linearly with dose of AFB1 and there were strong correlations at the individual rat level with both chromosomal aberrations (r = 0.92; p < 0.0001) and micronucleus frequency (r = 0.86; p < 0.0001). These data suggest that the AF-alb may reflect the level of genetic alteration resulting from the initial binding of this carcinogen to cellular DNA. Therefore, this adduct used as a biomarker in studies of human exposure to aflatoxin may provide information not only on exposure but also on the risk of genetic alterations consequent to that exposure.

Assessment of cytogenetic changes in human populations at risk in Egypt

Humans are exposed to numerous environmental agents that can increase the probability of mutagenicity and carcinogenicity. Most of environmental exposures involve concurrent or sequential exposure to several agents in air, water, and food. Interactive effects in carcinogenesis have been described for a certain number of combinations of agents. They are described in terms of enhancement or inhibition of carcinogenesis. Risk assessment of exposure to environmental agents can start either from laboratory studies after exposure to different agents or from epidemiological studies in relation to actual exposure. The use of genotoxicity testing is essential for assessment of potential human toxicity so that hazards can be prevented. Cytogenetic monitoring of human populations exposed to environmental agents has proved to be a useful tool for detecting their mutagenic effects. Cytogenetic analysis of human chromosomes in peripheral lymphocytes allows direct detection of mutation in somatic cells. Various methods can be used for chromosomal analysis (conventional chromosomal analysis, sister chromatid exchange, micronucleus frequency detection). Micronucleus frequency can be detected either in peripheral blood lymphocytes or in exfoliated cells. Different examples of human population studies are presented in this review. Several problems which are found in biomonitoring studies are discussed.