Changes in wheat plastid membrane properties induced by cadmium and selenium in presence/absence of 2,4-dichlorophenoxyacetic acid

The aim of the work was to recognize the effect of cadmium (Cd) and selenium (Se) onto properties of plastid lipid membranes. Plastids were isolated from wheat calli cultured during 2 weeks on Murashige–Skoog media with presence/absence of 2,4-dichlorophenoxyacetic acid. Plastids obtained in presence of 2,4-D represented an earlier developmental stage in comparison to those, got in absence of 2,4-D, which reached a pre-chloroplast stage. The studied metals were introduced to culture media separately (2 μM Na₂SeO₄ or 800 μM CdCl₂) or together (Se + Cd). The changes of following properties of plastid envelope membrane caused by both metals were measured: composition of main lipid fractions, their fatty acid saturation, membrane fluidity, lipid peroxidation and membrane zeta potential. Results of experiments led to the conclusion that galactolipid component plays a predominant role in modification of plastid membrane properties responding to Cd and Se addition. It was shown that galactolipid protecting reaction to Cd toxic action can consists in increased plastid envelope membrane stiffness. The presence of hormone (2,4-D) and Se did not counterbalance Cd toxic effects (at least under concentration level applied in the experiments). Se applied separately can probably stimulate plastid/chloroplast transformation in wheat cells by increasing a galactolipid unsaturation degree. The zeta potentials seem to be important physicochemical parameter in determination of properties of membranes exposed to metal stress conditions.     

Effects of Sodium Selenite and Germination on the Sprouting of Chickpeas (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and Its Content of Selenium,Formononetin and Biochanin A in the Sprouts

To improve the nutritional value of chickpea food, selenium (Se)-rich chickpea sprouts were produced by germination of chickpea seeds for 6 days at 28 centigrade in the presence of various concentrations of Na₂SeO₃ in germination solution. High concentrations of selenite were found to inhibit the growth of chickpea sprout and the biosynthesis of isoflavones formononetin and biochanin A. However, chickpea sprouts could tolerate up to ∼50 mg/L of Na₂SeO₃, under which condition the product chickpea sprouts contained a high Se content (2.14 μg/g dry weight) and a moderate high content of isoflavones (601.56 μg biochanin A/g dry weight and 578.11 μg formononetin/g dry weight). Se was incorporated in chickpea sprout in the form of selenomethionine. Thus, Se-enriched chickpea sprouts may serve as a convenient dietary source of Se and of isoflavones, including formononetin and biochanin A.     

Accumulation and volatilization of different chemical species of selenium by plants

Selenium (Se) removal from polluted waters and soils is especially complicated and highly expensive. Phytoremediation has been suggested as a low-cost, efficient technology for Se removal. Plants remove Se by uptake and accumulation in their tissues, and by volatilization into the atmosphere as a harmless gas. Unraveling the mechanisms of Se uptake and volatilization in plants may lead to ways of increasing the efficiency of the phytoremediation process. The objectives of this study were: (i) to determine the effect of different Se forms in the root substrate on the capacity of some plant species to take up and volatilize Se; (ii) to determine the chemical species of Se in different plant parts after the plants were supplied with various forms of Se; and (iii) to determine the influence of increasing sulfate levels on plant uptake, translocation, and volatilization of different Se species. Plants of broccoli (Brassica oleracea var. botrytis L.), Indian mustard (Brassica juncea L.), sugarbeet (Beta vulgaris L.) and rice (Oryza sativa L.) were grown hydroponically in growth chambers and treated for 1 week with 20 μM Se as Na₂SeO₄, Na₂SeO₃ or L-selenomethionine (SeMeth) and increasing sulfate levels. The data show that shoots of SeO₄-supplied plants accumulated the greatest amount of Se, followed by those supplied with SeMeth then SeO₃. In roots, the highest Se concentrations were attained when SeMeth was supplied, followed by SeO₃, then SeO₄. The rate of Se volatilization by plants followed the same pattern as that of Se accumulation in roots, but the differences were greater. Speciation analysis (X-ray absorption spectroscopy) showed that most of the Se taken up by SeO₄-supplied plants remained unchanged, whereas plants supplied with SeO₃ or SeMeth contained only SeMeth-like species. Increasing the sulfate level from 0.25 mM to 10 mM inhibited SeO₃ and SeMeth uptake by 33% and 15–25%, respectively, as compared to an inhibition of 90% of SeO₄ uptake. Similar results were observed with regard to sulfate effects on volatilization. We conclude that reduction from SeO₄ to SeO₃ appears to be a rate-limiting step in the production of volatile Se compounds by plants. Inhibitory effects of sulfate on the uptake and volatilization of Se may be reduced substantially if Se is supplied as, or converted to, SeO₃ and/or SeMeth rather than SeO₄. Received: 27 February 1998 / Accepted: 30 March 1998     

Effect of Selenium Pre-treatment on Antioxidative Enzymes and Lipid Peroxidation in Cd-exposed Suckling Rats

Since there are no data about the protective role of selenium (Se) against cadmium (Cd)-induced oxidative damage in early life, we studied the effect of Se supplementation on antioxidative enzyme activity and lipid peroxidation (through thiobarbituric acid reactive substances; TBARS) in suckling Wistar rats exposed to Cd. Treated animals received either Se alone for 9 days (8 μmol, i.e., 0.6 mg Se as Na₂SeO₃ kg⁻¹ b.w., daily, orally; Se group), Cd alone for 5 days (8 μmol, i.e., 0.9 mg Cd as CdCl₂ kg⁻¹ b.w., daily, orally; Cd group), or pre-treatment with Se for 4 days and then co-treatment with Cd for the following 5 days (Se + Cd group). Our results showed that selenium supplementation, with and without Cd, increased SOD activity in the brain and kidney, but not in the liver and GSH-Px activity across all tissues compared to control rats receiving distilled water. Relative to the Cd group, Se + Cd group had higher kidney and brain SOD and GSH-Px activity (but not the liver), while in the liver caused increased and in the brain decreased TBARS level. These results suggest that Se stimulates antioxidative enzymes in immature kidney and brain of Cd-exposed rats and could protect against oxidative damage.     

Arsenite-Induced Apoptosis Is Prevented by Selenite in A375 Cell Line

Arsenic trioxide induces apoptosis and clinical remission in patients diagnosed with acute promyelocytic leukemia. The human malignant melanoma A375 cells were treated with NaAsO₂ (0.1–130 μM) and also treated with combined 10 μM NaAsO₂ and 10 μM Na₂SeO₃. NaAsO₂ arrested cell growth in the G1 phase and induced apoptosis in a concentration- and time-dependent manner. In contrast, administration of Na₂SeO₃ antagonized the cell growth inhibition and apoptosis induced by NaAsO₂. The NaAsO₂ treatment resulted in a marked increase in p53 protein as early as 4 h and in Bcl-2 protein level by 12 h. In addition, p53 downregulation accompanied the combined treatment of NaAsO₂ and Na₂SeO₃. Thus, our results indicate upregulation of p53 and Bcl-2 play a crucial role in the NaAsO₂-induced G1 arrest and apoptosis of A375 cells and that downregulation p53 appears to contribute to the inhibition by Na₂SeO₃ of the effects induced by NaAsO₂.     

Selenium-Induced Up-Regulation of the Antioxidant Defense and Methylglyoxal Detoxification System Reduces Salinity-Induced Damage in Rapeseed Seedlings

The present study investigates the regulatory role of exogenous selenium (Se) in the antioxidant defense and methylglyoxal (MG) detoxification systems in rapeseed seedlings exposed to salt stress. Twelve-day-old seedlings, grown in Petri dishes, were supplemented with selenium (25 μM Na₂SeO₄) and salt (100 and 200 mM NaCl) separately and in combination, and further grown for 48 h. The ascorbate (AsA) content of the seedlings decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) increased with an increase in the level of salt stress, while the GSH/GSSG ratio decreased. In addition, the ascorbate peroxidase (APX) and glutathione S-transferase (GST) activity increased significantly with increased salt concentration (both at 100 and 200 mM NaCl), while glutathione peroxidase (GPX) activity increased only at moderate salt stress (100 mM NaCl). Glutathione reductase (GR) activity remained unchanged at 100 mM NaCl, while it was decreased under severe (200 mM NaCl) salt stress. Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, whereas a sharp decrease of these activities was observed under severe salt stress (200 mM NaCl). Concomitant increases in the levels of H₂O₂ and lipid peroxidation (MDA) were also measured. Exogenous Se treatment alone had little effect on the non-enzymatic and enzymatic components. However, further investigation revealed that Se treatment had a synergistic effect: in salt-stressed seedlings, it increased the AsA and GSH contents; GSH/GSSG ratio; and the activities of APX, MDHAR, DHAR, GR, GST, GPX, CAT, Gly I, and Gly II. As a result, addition of Se in salt-stressed seedlings led to a reduction in the levels of H₂O₂ and MDA as compared to salt stress alone. These results suggest that the exogenous application of Se rendered the plants more tolerant to salt stress-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

The effect of dietary selenium supplementation on cadmium absorption and retention in suckling rats

Selenium (Se) reduces cadmium (Cd) toxicity in adult animals, but its effects in newborn animals are still unknown. This study investigated Cd (as CdCl₂) absorption, distribution, and retention in suckling rats receiving oral Se supplementation (as Na₂SeO₃) in equimolar doses (8 μmol Cd and/or Se per kg b.w./day). Selenium was given either before and during Cd exposure (Se_pre + Cd group; pre-treatment group) or only during Cd exposure (Se + Cd group). Rats were treated from postnatal day (PND) 6–14 as follows: controls (H₂O, PND 6–14), Se (PND 10–14), Cd (PND 10–14), Se_pre + Cd (Se PND 6–14 + Cd PND 10–14) and Se + Cd (Se + Cd PND 10–14). Selenium supplementation, especially pre-treatment, decreased Cd levels in the blood, brain, liver and kidney of suckling rats. Selenium levels in plasma, brain, and kidney also decreased. These findings suggest that higher Se intake could efficiently reduce Cd retention during the suckling period.     

Comparative assessment of selenite (SeIV) detoxification to elemental selenium (Se0) by <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Bacillus licheniformis, B. subtilis, B. cereus, Bacillus pumilus and Exiguobacterium sp., which were resistant up to 20 mg Na₂SeO₃/ml in nutrient broth and 40 mg/ml on nutrient agar plates, were isolated from contaminated soil and water. They grew from 25 to 45°C and pH 5 to 9. They had multiple metal and antibiotic resistances. All strains reduced selenite (SeIV) to elemental selenium (Se0) aerobically with a maximum reduction of 97% by B. pumilus after 144 h with Na₂SeO₃ at 500 μg/ml.     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

Exogenous Selenium Pretreatment Protects Rapeseed Seedlings from Cadmium-Induced Oxidative Stress by Upregulating Antioxidant Defense and Methylglyoxal Detoxification Systems

The protective effect of selenium (Se) on antioxidant defense and methylglyoxal (MG) detoxification systems was investigated in leaves of rapeseed (Brassica napus cv. BINA sharisha 3) seedlings under cadmium (Cd)-induced oxidative stress. Two sets of 11-day-old seedlings were pretreated with both 50 and 100???M Se (Na₂SeO₄, sodium selenate) for 24?h. Two concentrations of CdCl₂ (0.5 and 1.0?mM) were imposed separately or on the Se-pretreated seedlings, which were grown for another 48?h. Cadmium stress at any levels resulted in the substantial increase in malondialdehyde and H₂O₂ levels. The ascorbate (AsA) content of the seedlings decreased significantly upon exposure to Cd stress. The amount of reduced glutathione (GSH) increased only at 0.5?mM CdCl₂, while glutathione disulfide (GSSG) increased at any level of Cd, with concomitant decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) increased significantly with increased concentration of Cd (both at 0.5 and 1.0?mM CdCl₂), while the activities of glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at moderate stress (0.5?mM CdCl₂) and then decreased at 1.0?mM severe stress (1.0?mM CdCl₂). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon exposure to any levels of Cd. Selenium pretreatment had little effect on the nonenzymatic and enzymatic components of seedlings grown under normal conditions; i.e., they slightly increased the GSH content and the activities of APX, GR, GST, and GPX. On the other hand, Se pretreatment of seedlings under Cd-induced stress showed a synergistic effect; it increased the AsA and GSH contents, the GSH/GSSG ratio, and the activities of APX, MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II which ultimately reduced the MDA and H₂O₂ levels. However, in most cases, pretreatment with 50???M Se showed better results compared to pretreatment with 100???M Se. The results indicate that the exogenous application of Se at low concentrations increases the tolerance of plants to Cd-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

Root morphology, root-hair development and rhizosheath formation on perennial grass seedlings is influenced by soil acidity

Cadmium (Cd) exerts a detrimental effect on the metabolism of plants, whereas selenium (Se) may protect them against various stressors through its antioxidative activity. In this in vitro study we investigated the impact of Se (2 µM Na₂SeO₄) on the growth, nutrient (P, S, K, Ca, Mg, B, Mn, Fe and Zn) concentrations and cell integrity of rape (Brassica napus oleifera) and two wheat (Triticum aestivum) genotypes subjected to Cd stress (600 µM CdCl₂). Rape accumulated both Cd and Se more than did wheat. In all plants, Cd markedly reduced the biomass, enhanced lipid peroxidation and diminished plasmalemma fluidity. A drop in the K uptake and the reduced plasmalemma permeability diminished the K efflux from the leaf cells. In contrast, Cd elevated S concomitantly with Zn, indicating an activity of detoxifying SH groups and SOD isoenzymes. When added alone, Se promoted the growth of all plants, it enhanced the accumulation of S, but the impact on other nutrients remained minor. In Cd-stressed plants, Se tended to counterbalance the Cd-induced changes in nutrients, it also reduced the lipid peroxidation and exerted positive effects on the cell membrane stability. The Cd stress and the protective role of Se were most evident in rape. The Finnish wheat genotype was less tolerant to Cd than the Polish one.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of brown oak (<Emphasis Type="Italic">Quercus semecarpifolia</Emphasis> Sm.) from seedling explants

An efficient and reproducible method for the regeneration of multiple shoots of brown oak (Quercus semecarpifolia Sm.) has been developed in which a part of the petiolar tube containing a primary shoot is used as the explant. Explants derived from in vitro grown seedlings were cultured either on Murashige and Skoog or Woody Plant medium (WPM) containing different concentrations of benzyladenine (BAP) throughout the range of 1–20 μM. WPM supplemented with 20 μM BAP was found to be best for adventitious shoot induction and for the multiplication of individual shoots. In-vitro-produced shoots were rooted using a two-step method. Firstly, shoots were cultured on WPM containing indolebutyric acid (IBA) at either 50 or 100 μM for 24 or 48 h. Secondly, the shoots were transferred to plant-growth-regulator-free half-strength WPM. The second step not only considerably improved the rooting percentage but also minimized the formation of basal callus. The most effective first-step treatment was found to be 100 μM IBA for 24 h, which initiated rooting at a frequency of 100%. Well-rooted plants were transferred to plastic cups containing nonsterile, sieved soil and farmyard manure, hardened under greenhouse conditions, and then successfully established in pots. This procedure is suitable for use in large-scale production of plants and may have potential application in additional oak species.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

In vitro seed germination and cultivation of the aromatic medicinal <Emphasis Type="Italic">Salvia stenophylla</Emphasis> (Burch. ex Benth.) provides an alternative source of α-bisabolol

The aromatic medicinal plant Salvia stenophylla contains α-bisabolol, making this plant an important contributor to the aromatherapy and cosmetic industries in South Africa. Due to its commercial importance, the cultivation of this plant using an in vitro system was considered. Firstly, seedlings were raised in vitro after breaking dormancy with light, smoke-water or chemical scarification treatments. Germination improved when seeds were smoke-treated or soaked in 70% (v/v) H₂SO₄. Vigorous plantlet regeneration was achieved when seedling explants were cultured on Murashige and Skoog (1962) medium with 5.7 μM IAA and 8.9 μM BA. The potential regeneration capacity for this protocol was estimated and over 1,000 plantlets can be produced from a single shoot (6.67 cm with 4–6 nodes) over a period of 3 months. Plants rooted easily regardless of their growth medium. This was followed by their successful rapid establishment and normal growth out of culture (75%). Finally, the volatile compounds in in vitro plants were compared to ex vitro plants via headspace solid phase microextraction linked to gas chromatography–mass spectrometry. The chemical complexity of microplants was similar to wild plants with in vitro plants continuing to produce α-bisabolol (21%) at high levels.     

High efficiency <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from epicotyl explants of Chinese peanut cultivars

An efficient organogenesis and micropropagation system was developed for in vitro plant regeneration of multiple cultivars of peanut (Arachis hypogaea). The system was used to regenerate plants from nine cultivars: Luhua no. 9, Luhua no. 13, Luhua no. 14, Fenghua no. 1, Fenghua no. 3, Fenghua no. 5, Huayu no. 23, Qinglan no. 2, and Baisha 1016. Epicotyl and embryo axis explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 33.29–44.39 μM 6-benzyladenine (BAP) and 2.15–4.30 μM α-naphthaleneacetic acid (NAA). The highest rate of shoot formation was observed in epicotyl explants taken from 4-d-old seedlings (5.1 ± 1.4 shoots per explant). Optimum shoot development was observed in explants cultured on MS medium containing 4.48 μM BAP and 2.89 or 5.78 μM gibberellin (GA₃). Well-developed shoots (3–5 cm high) formed roots after 2 wk on MS medium containing 0–2.69 μM NAA. We observed that all multiple shoots formed at the site of epicotyl incision and at the upper end of each section, indicating physiological polarity of shoot formation. The maximum shoot induction rate for Luhua no. 9, Luhua no. 13, Luhua no. 14, Fenghua no. 1, Fenghua no. 3, Fenghua no. 5, Huayu no. 23, Qinglan no. 2, and Baisha 1016 was 60.0%, 83.3%, 80.7%, 91.5%, 86.0%, 59.7%, 75.0%, 67.3%, and 72.7%, respectively. This regeneration system will play a vital role in achieving the genetic improvement of Chinese peanut.     

In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

In vitro differential effects of sodium selenite on the growth of human hepatoma cells and human embryonic liver cells

The effects of various concentrations of Na₂SeO₃ on human hepatoma cells and human embryonic liver cells was investigated in vitro. For human hepatoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL Na₂SeO₃, mitotic activity of human hepatoma cells were partially arrested. In human embryonic liver cells continuously treated with Na₂SeO₃, (1 μg/mL) cell count of the treated group decreased only by d 7; mitotic index, labeled index, and mean silver grain number per 50 labeled nuclei were the same as in the control group on exposure to 1, 3, and 5 μg/mL for up to 72 h. In mixed cultures of human hepatoma and embryonic liver cells treated with 3 and 5 μg/mL of Na₂SeO₃ for 24 h, hepatoma cells showed vacuolated cytoplasms, distorted nuclei, condensed chromatin, and even pyknosis, whereas the embryonic liver cells retained a normal morphology under the same treatment.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Effect of selenium compounds on the damage induced by oxysterol on rat arterial walls

Wistar rats were fed Se-deficient (0.017±0.002 mg Se/kg) and Seadequate (0.32±0.045 Se mg/kg) diets for 12 mo and then were given 5 mg/kg of cholestane-3β,tα,6β-triol (3-triol), intravenously. Se compounds (Na₂SeO₃ and ebselen) were supplemented in different doses and times to the Se-deficient rats. Twenty-four hours after 3-triol infusion, the changes in ultrastructures of rat aorta were examined by scanning electron micrography (SEM) and transmission electron micrography (TEM). SEM examinations showed that 3-triol induced diffused injuries on arterial endothelial urfaces of long-term Se-deficient rat, and a large number of holes or craterlike defects were observed. TEM examinations further showed that 3-triol induced swelling, necrosis, and shedding of endothelial cells, which resulted in the destruction of endothelial integrity. Mean-while, smooth muscle cells proliferated and migrated toward intimae; the breakage of internal elastic lamina benefited the migration of smooth muscle cells. Supplemented with Na₂SeO₃ (40 μg/kg, 10 d per continuum) and ebselen (20 mg/kg), respectively, exhibited significant protection from damages induced by 3-triol. It seems that protecting mechanisms were different between Na₂SeO₃ and ebselen. The present investigation gave visual evidence that both injuries induced by cholesterol oxides and the Se nutritional status contributed to the development of atherosclerosis.