Molecular characterization of hereditary persistence of foetal haemoglobin mutation by restriction fragment length polymorphism mapping.

Characterization of hereditary persistence of foetal haemoglobin (HPFH) mutation in a family from West Bengal, India, was carried out by analysing the structure of the 5'-Ggamma-Agamma-psibeta-delta-beta-3' globin gene region by using the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique. The HPFH in this family was due to a deletion in the beta-globin gene cluster spanning at least from the Hin cII/5' psibeta to the Hin fI/3' beta RFLP site. This work indicates the importance of RFLP-PCR technique in characterization of the HPFH mutation.

A review on the origin and spread of deleterious mutants of the beta-globin gene in Indian populations.

Deleterious mutations of the human beta-globin gene are responsible for beta-thalassaemia and other haemoglobinopathies, which are the most common genetic diseases in Indian populations. A highly heterogeneous distribution of those mutations is observed in India and certain mutations are restricted to some extent to particular groups only. The reasons behind the geographical clustering and origin of the mutations in India is a highly debated issue and the evidence is conflicting. Our present article aims at tracing the origin of the deleterious beta-globin mutation and evaluates the role of different evolutionary forces responsible for the spread and present distribution of those mutations in Indian populations, using data from molecular biology and statistical methods. Mutations are generated essentially randomly, but "hot-spot" sites for mutation are reported for the beta-globin gene cluster, indicating sequence dependency of mutation. A single origin of a deleterious beta-globin mutation, followed by recombination (in a hot spot region) and/or interallelic gene conversion (within beta-globin gene) through time is the most plausible hypothesis to explain the association of those mutations with multiple haplotype backgrounds and frameworks. It is suggested that India is the place of origin of HbE and HbD mutations and that they dispersed to other parts of the would by migration. HbS mutants present in Indian populations are not of Middle East origin but rather a fresh mutation is the probable explanation for the prevalence among tribal groups. beta-thalassaemia represents a heterogeneous group of mutant alleles in India. Five common and twelve rare mutations have been reported in variable frequencies among different Indian populations. Gene flow of those mutant alleles from different populations of the world by political, military and commercial interactions possibly accounts for the heterogenous nature of beta-thalassaemia among Indians. A multiple allelic polymorphic system of the beta-globin gene exists in different populations. Dynamic interaction of the mutant alleles in the presence of different selective forces including falciparum malaria and biosocial patterns of Indian populations is discussed in order to explain the variable distribution and maintenance of those mutant alleles.

Chromosome damage induced by selenium salts in human peripheral lymphocytes.

Two inorganic salts of selenium, sodium selenite (Na(2)SeO(3)) and sodium selenate (Na(2)SeO(4)), were screened for damage to chromosome and cell division following exposure to human lymphocyte cultures. In vitro exposure of human peripheral blood lymphocytes to high concentration of two inorganic salts of selenium-sodium selenite (2.9 x 10(-5) M) and sodium selenate (2.65 x 10(-5) M)-was found to be lethal; no blast cells being formed. Lower concentrations of both salts, 5.8 x 10(-6) M and 1.06 x 10(-5) M, respectively, were highly mitostatic. Lower concentrations of sodium selenite (2.9 x 10(-6) M, 1.16 x 10(-6) M and 2.32 x 10(-7) M) and sodium selenate (5.3 x 10(-6) M, 2.65 x 10(-6) M and 1.06 x 10(-6) M), respectively, induced chromosomal aberrations and reduced cell division in proportions directly related to the dose. Sodium selenite was considerably more clastogenic than sodium selenate.

Dietary protection by iron against clastogenic effects of short-term exposure to arsenic in mice in vivo.

Iron, as freshly prepared aqueous solution of ferrous sulfate, was administered by gavage to laboratory bred Swiss albino mice. The concentration used was 152 mg/kg body weight (1/10 of the LD(50)). While screening for protection against arsenic, in one set of experiment exposure to iron was followed after 2 hr by gavaging with 2.5 mg/kg body weight (1/10 of the LD(50)) of arsenic as sodium (III) meta arsenite in distilled water. In another set, equal amounts (1:1) of ferrous sulfate and sodium arsenite were administered simultaneously. Control sets were given sodium m-arsenite alone and distilled water (vehicle). After exposure for 24 hr in all experiments, mice were sacrificed and chromosome preparations were made from bone marrow according to a colchicine-hypotonic-fixation-air-drying-Giemsa schedule. Cytogenetic endpoints screened were chromosome aberrations and divisional frequencies. Sodium arsenite alone was highly clastogenic. Ferrous sulfate, whether given together with or before exposure to sodium arsenite, reduced the clastogenic effects of the latter to a significant extent.

Clastogenic effects of zinc chloride on human peripheral blood leucocytes in vitro.

The clastogenic effects of three different concentrations of zinc chloride on human peripheral blood leucocytes were studied in vitro. The highest concentration (1.5 x 10(-3) M) was lethal after 48 and 72 h of culture and no blast cells were formed. The two lower concentrations (3.0 x 10(-4) M and 3.0 x 10(-5) M) significantly reduced the frequency of cell division, induced chromatid breaks and damaged cells in frequencies significantly higher than in control experiments maintained in sodium chloride and in distilled water.

Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels.

study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents.

Comparison of chromosome damage induced by three zinc compounds using human leukocyte culture.

The degree of chromosome damage induced by three compounds of zinc (zinc chloride, zinc sulfate, zinc acetate) was compared in human leucocytes in vitro. Three concentrations of each salt, 3.0 x 10(-5)M, 3.0 x 10(-4)M, and 1.5 x 10(-3)M, were added to leukocyte cultures. The cells were harvested after 48 and 72 h and chromosome spreads were prepared following a colchicine-hypotonic-fixation-air drying-Giemsa staining schedule. The end point screened was chromosome aberrations. All three salts were lethal at the highest concentration. The degree of chromosome damage was directly proportional to the concentrations used for zinc sulfate and zinc acetate but not for zinc chloride.

Protection against cytotoxic effects of arsenic by dietary supplementation with crude extract of Emblica officinalis fruit.

Dietary administration of a crude aqueous extract of Emblica officinalis Gaertn. fruit reduced significantly the cytotoxic effects of sodium arsenite administered orally. The crude extract (685 mg/kg bw) was given daily by gavaging to age and sex matched laboratory bread Swiss albino mice for 7 and 14 days, followed by a single dose of sodium arsenite (2.5 mg/kg bw = 1/10 of LD(50)). The animals were killed after 24 h and chromosome preparations made following a schedule of colchicine-fixative-air drying-Giemsa. The endpoints screened were chromosomal aberrations and damaged cells. The crude extract reduced arsenic damage bringing the cells almost to the normal level.

Prevention of cytotoxic effects of arsenic by short-term dietary supplementation with selenium in mice in vivo.

Interaction between selenium and arsenic has been used to protect against the genotoxic effects of sodium arsenite through dietary intervention by an equivalent amount (1/10 LD50) of sodium selenite. The two salts were administered by gavaging to laboratory bred Swiss albino mice sequentially and in combination. Cytogenetic endpoints, including chromosomal aberrations (CA) and damaged cells (DC) were recorded 24 h after exposure from chromosome spreads in bone marrow cells. Administration of sodium selenite 1 h before sodium arsenite reduced the clastogenic effects of the latter significantly. The protection was less when the salts were given together and negative when arsenite was given before selenite. Histological changes were recorded. Such reduction of arsenic toxicity through dietary intervention by selenium is of significance in protecting against the widespread toxicity observed in human populations exposed to arsenic through drinking water from contaminated deep tubewells in West Bengal and Bangladesh.

Comparison of clastogenic effects of inorganic selenium salts in mice in vivo as related to concentrations and duration of exposure.

Inorganic selenium compounds in the diet have been known to protect against cancer in laboratory animals, but were harmful in high concentrations. In the present work, the relative effects of two salts, sodium selenite and sodium selenate, administered to mice in vivo, in different concentrations and durations of exposure, were compared. Aqueous solutions of each salt (7, 14, 21 and 28 mg Kg-1 bw) were fed by gavaging to mice matched in age and sex. The animals were sacrificed at intervals of 6, 12, 18 and 24 h and chromosome preparations were made following the usual schedule of colchicine-hypotonic-fixative-airdrying-Giemsa staining. The endpoints screened were chromosomal aberrations (CA) and damaged cells (DC). Both salts affected chromosome structure and spindle formation, sodium selenite being more cytotoxic than sodium selenate. The frequencies of aberrations induced were directly proportional to the concentrations used and duration of exposure.

Inhibition of clastogenic effects of arsenic through continued oral administration of garlic extract in mice in vivo.

Crude aqueous extract of garlic bulbs (Allium sativum L. single clove variety) was administered by gavage to mice of both sexes daily for up to 30 and 60 days, in doses corresponding to 6 g for a 60 kg human body. Sodium arsenite (at 1/50 of LD50 dose) was injected subcutaneously to mice on every 7th day of the experiment. Chromosome preparations made from bone marrow following flame drying Giemsa schedule were screened for chromosomal aberrations. The clastogenic affects of prolonged exposure to sodium arsenite --a strong clastogen-- was reduced by a highly significant amount when crude garlic extract, in the dose used, was given daily to the mice by intubation for the same period.

The occurrence of beta-thalassemia mutation and its interaction with hemoglobin E in the eastern India.

The distribution of variant hemoglobin in India has been related to various ethnic groups among other factors. beta-Thalassemia is the most frequent monogenic disorder in the country. Analysis of hemoglobin of 435 cases from Eastern India was performed by electrophoresis and by other quantitative methods. Analysis of the beta-globin gene of 112 cases used ARMS (amplification refractory mutation system)-PCR (polymerase chain reaction) techniques showing that IVS-1 nt 5 (G-->C) is the most prevalent mutation in populations from Eastern India. IVS-1 nt 5 (G-->C) interacts with the codon 26 (G-->A) mutation to produce E beta-thalassemia phenotype in the samples from West Bengal, India.

Selenium salts and chromosome damage.

Sodium selenite and sodium selenate, fed by gavaging to age-matched male Swiss albino mice and observed after 24 h following a colchicine-fixative-air drying-Giemsa schedule, were found to induce chromosome breaks and spindle disturbances in bone marrow cells. The four concentrations used were fractions of LD50 and the effects were directly proportionate to the concentration of the chemical. Sodium selenite induced a slightly higher frequency of chromosomal aberrations than sodium selenate.

Dietary chemoprevention of clastogenic effects of 3,4-benzo(a)pyrene by Emblica officinalis Gaertn. fruit extract.

Dietary supplementation with extract of fruit of Emblica officinalis Gaertn. (a rich source of vitamin C) to mice in vivo significantly reduced the cytotoxic effects of a known carcinogen, 3,4-benzo(a)pyrene. Age-matched Swiss albino mice were fed by gavaging the fruit extract daily for 28 days. From day 9, one dose of the carcinogen was given on alternate days up to a total of eight doses. On day 29, all mice were transferred to normal diet. Control sets received the extract alone, the carcinogen alone and olive oil alone. All mice were sacrificed at 12 weeks and 14 weeks after the end of the experiment. Chromosome preparations were made from bone marrow after the usual colchicine-hypotonic-fixative-air drying-Giemsa staining schedule. Cytogenetic end points screened were the frequencies of chromosomal aberrations and of damaged cells induced. The cytotoxic effects were significantly lower in the mice given the fruit extract with the carcinogen than in those given the carcinogen alone.

Chlorophyll and chromosome breakage.

Increased consumption of green vegetables in the diet has been associated with protection against carcinogenic effects and related mutagenic and clastogenic (chromosome breaking) activity of genotoxic agents. Chlorophyll, present in all green plant parts, has been suggested to be a major protective factor in the process. We have, however, observed that while a crude aqueous extract of Indian spinach leaf significantly reduced genotoxic effects, chlorophyll alone was ineffective. On the other hand, chlorophyll, both as an aqueous extract from the leaf and in a purified commercial form, induced a significantly high frequency of chromosome breaks in bone marrow cells of mice on oral administration. The crude aqueous extract of the leaf was non-toxic. The protective activity of the crude leaf extract may be attributed to the total effect of the interaction between different components, in which the clastogenicity of chlorophyll has been neutralized.

Dietary garlic extract in modifying clastogenic effects of inorganic arsenic in mice: two-generation studies.

Mice are fed by gavage crude garlic extract (100 mg/kg b.wt.) for 30 consecutive days. One set was administered sodium arsenite (0.1 mg/kg b.wt.) simultaneously. Another set was treated with sodium arsenite only. Mice given distilled water were kept as negative control. Exposed mice from each set were sacrificed and bone marrow preparations examined for chromosomal aberrations and damaged cells. Sodium arsenite is a strong clastogen and the effects were reduced to a significant level by prolonged administration of garlic extract. For F1 studies, exposed male mice were mated with exposed female mice, and the progeny examined. In the progeny, clastogenic effects of sodium arsenite persisted in a lower degree, indicating that the metal is able to cross the transplacental barrier. There was no statistically significant difference between the effect in progeny of parents only given sodium arsenite when given simultaneously for prolonged periods in the parents; however, the effect is meagre in the next generation.

Clastogenic activity of pure chlorophyll and anticlastogenic effects of equivalent amounts of crude extract of Indian spinach leaf and chlorophyllin following dietary supplementation to mice.

Dietary consumption of green vegetables has been associated with protection against mutagenic and clastogenic activity of genotoxicants. Chlorophyll, being present in all green plants, had earlier been suggested to be the principal factor involved. Mice were administered (i) crude aqueous extract of leaf of Indian spinach, Beta vulgaris L. var.benghalensis Hort., and equivalent amounts of (ii) chlorophyll extracted from the leaf; (iii) purified chlorophyll, (iv) chlorophyllin, a sodium-copper derivative of chlorophyll; daily for 7 days. On day 7, one set of mice from each treatment was administered potassium dichromate-a known metallic clastogen. The mice were sacrificed after 24 hours. Chromosome preparations were made from bone marrow following the usual colchicine-air dry-Giemsa schedule. The cytogenetic endpoints scored were chromosomal aberrations and damaged cells. Crude leaf extract and chlorophyllin were nonclastogenic and reduced the clastogenic effects of potassium dichromate to the control distilled water level. Chlorophyll alone, whether extracted from the leaf or obtained in commercially purified form, was clastogenic and could reduce the effects of the chromium salt only to its own level. The protective action of the crude leaf extract may be attributed to the total effect of the interaction between the different components within the leaf extract, in which the clastogenicity of chlorophyll had been neutralized.

Effects of crude garlic extract on mouse chromosomes in vivo.

Three concentrations (25, 50 and 100 mg/kg body weight) of fresh garlic (Allium sativum L.) were administered daily by gavage to Swiss albino mice for different durations up to 60 days. These concentrations had been observed to protect significantly against effects of known clastogens. The endpoints scored were frequencies of chromosomal aberrations and damaged cells induced in bone marrow preparations. These parameters were found to be directly dose dependent and after an initial enhancement at 7 days, were reduced following prolonged exposure for 30 and 60 days to the low level observed at 24 hr. Therefore, administration of a low concentration of garlic extract daily is suggested for at least 30 days to obtain the maximum benefit of the extract in protecting against the clastogenic effects of known genotoxicants.