DNA damage-induced by sodium flouride (NaF) and the effect of cholicalciferol

It is known that the high electronegativity of fluorine affects various soft tissues, especially the bone structure in organisms. Of these tissues are the kidneys, which play an important role in the excretion of fluoride from the body. Fluoride affects many cellular mechanisms. One of these effects is DNA damage. Our study aimed to investigate the likely protective effect of cholecalciferol (vitamin D3) on genomic DNA damage-induced NaF depending on concentration and time. The IC25 and IC50 values of NaF for 3, 12 and 24 h and optimum dose of increase in proliferation to vitamin D₃ through MTT assay in NRK-52E kidney cells were determined. DNA damage was significantly increased (p < 0.05) compared to the control group in all groups except for vitamin D₃. It was determined that treatment with NaF together with vitamin D₃ decreased the DNA damage compared to NaF treated groups for 3 and 12 h. NaF combined with vitamin D3 was determined statistically to decrease (p < 0.05) DNA damage compared to NaF treated groups for 24 h. As a result, it was determined that the treatment with cytotoxic concentration NaF depending on the time significantly increased (p < 0.05) the genomic DNA damage, but NaF treatment together with vitamin D₃ decreased the DNA damage in renal cells depending on the time. It was concluded that vitamin D₃ may be useful in preventing DNA damage caused by NaF.     

Visualization of cellular DNA crosslinks by atomic force microscopy

Cellular DNA crosslinks are a type of DNA damage induced by toxic chemicals or high‐energy radiation. If damaged DNA is not rapidly repaired, cells will die or mutate. To evaluate the types of DNA damage and their influence on vital cell activities, it is necessary to be able to detect DNA crosslinks. To date, indirect methods such as alkaline elution, potassium chloride–sodium dodecyl sulfate assay and comet assay have been used to detect DNA damage. Direct morphological observation, on the other hand, may be a useful tool to differentiate the types of DNA damage. In this report, atomic force microscopy (AFM) has been employed to visualize the breakage and crosslinking of cellular DNA strands in cells treated with formaldehyde and hydrogen peroxide. Our results showed that toxic chemical‐induced crosslinking of cellular DNA occurred in a dose‐dependent manner. DNA conglomerates were observed with high concentrations of formaldehyde, and the AFM observations were consistent with those of a comet assay. Our experiments demonstrate that AFM is an efficient method to differentiate the types of DNA damage. SCANNING 31: 75–82, 2009. © 2009 Wiley Periodicals, Inc.     

Application of confocal laser scanning microscopy to analysis of H2O2-induced DNA damage in human cells

Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in lowmelting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting “comets” provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive and reproducible quantitation of single-stranded breaks of cellular DNA.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen–glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study, we investigated the protective effect of hyperbaric oxygen (HBO) on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism. PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established. Cells were divided into a control group, model group, hyperbaric air (HBA) group and HBO group. The cell viability was detected by the CCK8 assay. Hoechst 33342 and PI staining assays and mitochondrial membrane potential (MMP) assays were used to detect cell apoptosis. The ultrastructure of cells, including autophagosomes, lysosomes, and apoptosis, were examined using a transmission electron microscope. The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry. Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion. HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygen-glucose deprivation/reperfusion. Importantly, we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway. In this study, the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1, p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased, while the levels of LC3B, Atg5 and p53 increased. However, after HBO treatment, these autophagy-related indexes were reversed. In addition, observation of the cell ultrastructure with transmission electron microscopy found that in the model group, a significant increase in the number of autophagic vesicles was observed. In the HBO group, a decrease in autophagic vesicles was observed. The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.     

Deconstructing the molecular mechanisms of cell cycle control in a mouse adrenocortical cell line: roles of ACTH

This is a progress report of an attempt to deconstruct the signaling network underlying cell cycle control in the mouse Y1 adrenocortical cell line, aiming to uncover ACTH growth regulatory pathways. Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene. Despite this oncogenic lesion, Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the sequential events comprising the mitogenic response triggered by FGF2 in G0/G1-arrested Y1 cells: 1) activation of ERK1/2 and PI3K, by 5 minutes; 2) induction of c-Fos and c-Myc proteins by 2 hours; 3) induction of cyclin D1 protein by 5 hours; 4) phosphorylation of Rb protein between 6 and 8 hours; 5) onset of DNA synthesis by 8-9 hours. In this cell line, ACTH-receptor (ACTH-R) activates contradictory pathways of growth regulation. First, ACTH coordinately induces fos and jun gene families via activation of both ERK1/2 and cAMP/PKA pathways, resembling a mitogen. Second, ACTH-R triggers cAMP/PKA-mediated antimitogenic mechanisms comprised of Akt/PKB dephosphorylation/deactivation, c-Myc protein degradation, and p27(Kip1) protein induction. Induction of cyclin D1 depends on activation of both ERK1/2 and PI3K, but is not affected by ACTH action. As a consequence, ACTH antagonizes FGF2 mitogenic activity but ectopic expression of the c-Myc protein (via MycER fusion protein) is sufficient to abrogate this ACTH antagonistic effect over FGF2 mitogenic activity. Ectopic expression of both c-Myc and cyclin D1 is not sufficient to drive G0/G1-arrested Y1 cells into S phase, but when the sustained expression of these two proteins is complemented by ACTH treatment it promotes G1 phase progression and DNA synthesis initiation. In conclusion, ACTH-receptor lacks signaling potential sufficient to initiate a mitogenic response in Y1 adrenocortical cells and, therefore, cannot substitute for bona fide mitogens like FGF2.     

DTL facilitates the Fanconi anemia pathway for ultraviolet-induced DNA repair in retinal pigment epithelial cells

The excessive energy of light, especially the invisible rays with lower wavelength, is basically absorbed by retinal pigment epithelium (RPE) and usually causes DNA damage. The molecular mechanism behind DNA damage repair response to this frequent stress in RPE is not clearly understood. In this study, we determined that the Fanconi anemia (FA) pathway was activated in human RPE ARPE-19 cells after ultraviolet (UV) B and C treatment. Moreover, immunoprecipitation (IP) of FANCD2 indicated that denticleless E3 ubiquitin protein ligase homolog (DTL) closely interacted with FANCD2. Knockdown of DTL weakened the activity of the FA pathway in ARPE-19 cells responding to UV treatment. Finally, the DTL promoter was incubated with a biotin-labeled probe and pulled down by streptavidin beads followed by the genomic DNA sonication. p53 was indicated by mass spectrum and further determined by chromatin IP assay. Taken together, our results demonstrated that DTL regulated by p53 could activate the FA pathway for UV-induced DNA damage repair in retinal pigment epithelial cells.     

Programmed neuronal cell death induced by HIV-1 tat and methamphetamine

Apoptosis and autophagy are the two major types of programmed cell death (PCD) in neurons. Homeostatic autophagy often precedes apoptosis, and when apoptosis is blocked, the failure to keep homeostasis will lead to necrosis instead. It has been reported that human immunodeficiency virus (HIV) infected methamphetamine (Meth) abusers represent greater neuropathological abnormalities than Meth abusers or HIV-positive non-Meth users. Recent publications suggest that Tat and Meth when administered together result in greater neuronal damage than when administered separately. However, the cellular events of the combined Tat-Meth effect have not yet been fully characterized. Therefore, we investigated the effects of Tat and/or Meth on apoptosis and autophagy to elucidate whether PCD was involved in Tat and/or Meth-induced neuronal damage. Annexin-V-FITC/PI staining assay was used to detect cellular apoptosis using a neuroblastoma cell line SH-SY5Y. Cellular ultrastructural changes were observed under transmission electron microscopy (TEM). Flow-cytometric data showed apoptosis following Meth treatment, and more extensive apoptosis with Tat + Meth treatment. The most important finding was that the autophagosome and/or multilamellar bodies (MLBs) were most pronounced with Tat + Meth treatment, were less so with Meth treatment, and infrequent with Tat treatment. This suggests the involvement of autophagy and apoptosis in Tat with Meth-elicited cell damage. However, the relation between apoptosis and autophagy remains unknown in this experiment. Further research is needed to analyze the relation among related molecules. A thorough understanding of this multifaceted relationship will be critical for the assessment of therapeutic modalities for patients with HIV with drug abuse.     

Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma (SH-SY5Y) cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines. The human neuroblastoma cells (SH-SY5Y) were exposed to the phosphatase inhibiting toxin okadaic acid (OA) or hydrogen peroxide (H2O2) to induce apoptosis as well as generate reactive oxygen species (ROS). Mitoxantrone (MXT) was used as a positive control for apoptosis. The SH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide (HyPer). The ERp57, also called GRP58 (Glucose-regulated protein 58), is a stress protein induced in conditions like glucose starvation and viral infection. Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells. ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death. ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy. These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.     

尼莫司汀和卡莫司汀导致细胞中DNA股间交联的高效液相色谱-电喷雾质谱联用研究

氯乙基亚硝基脲(CENUs)是临床常用的烷化剂类抗癌药物,主要通过导致DNA股间横向交联(dG-dC交联)发挥其抗癌作用。为建立高灵敏度的细胞中交联物的检测方法,评价不同种类CENUs的抗癌活性及耐药性,本研究使用尼莫司汀(ACNU)和卡莫司汀(BCNU)对具有不同O⁶-烷基鸟嘌呤-DNA-烷基转移酶(AGT)活性的NIH/3T3和L1210两种细胞进行药物处理,利用高效液相色谱-电喷雾串联质谱 (HPLC-ESI-MS/MS) 联用技术对经不同浓度药物处理后细胞中的dG-dC交联进行定量分析。该方法的检测限(S/N=5)和定量限(S/N=17)分别达到2 fmol和8 fmol,回收率为92.5%~107.4%,灵敏度和准确性均满足定量分析的要求。结果表明：ACNU导致的dG-dC交联率高于BCNU;经相同浓度的同种药物作用后,L1210细胞中交联率显著高于NIH/3T3细胞。这可为新型CENU类烷化剂的抗癌活性评价提供可靠的实验方法。     

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst‐33342, 4’, 6‐diamidino‐2‐phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin‐V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.     

Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP.

OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.     

Effect of time of restriction on the decrease in mitochondrial H2O2 production and oxidative DNA damage in the heart of food-restricted rats

In the present study, the question if medium-term (4 months) caloric restriction (40%) decreases mitochondrial H2O2 production and oxidative DNA damage was investigated. Caloric restriction (CR) is the only experimental manipulation that increases maximum life span. Previous long-term CR studies have showed that CR decreases the mitochondrial rate of free radical production in diverse tissues and species. Those studies agree with the idea that the superior longevity of the restricted animals can be partly due to their lower mitochondrial rate of free radical generation. However, caloric restriction effects strongly depend on implementation time. Previous studies have shown that the decrease induced by CR on oxygen radical generation and oxidative damage to mitochondrial DNA occurs after 1 year but not after 6 weeks of restriction. In the present investigation, mitochondrial H2O2 production did not change in medium-term (4 months) caloric restricted animals, and, in agreement with that, no differences were found in either mitochondrial or nuclear oxidative DNA damage between restricted and ad libitum-fed animals. These results confirm the importance of the time of CR implementation, and show that time longer than 4 months is needed to decrease the mitochondrial rate of free radical generation and the oxidative damage to mtDNA in the rat heart.     

有机金属钌抗癌药物与G-四链体DNA的相互作用研究

Guanine-rich sequence TTAGGG can form G-quadruplex(G4) at the end of human telomeres, which protect chromosomal ends from unwanted recombination and degradation and inhibit the activity of telomerase enzyme, The telomerase was shown to be active in 85%-90% of human cancer cells, but inactive in healthy and somatic cells. DNA is the potential target of organometallic ruthenium(II) anticancer complexes. It is of great importance to study the interaction of ruthenium complex with the senior structural DNA G4. The present work focused on investigating the interactions between organometallic ruthenium complexes and G-quadruplex. The pilot studies show that NH4^＋ can stabilize the G4 structure to reduce the binding of biphenyl ruthenium complexes to guanine bases, but the inhibitory effects disappeared as the concentration of ruthenium complexes increases. This result suggests that the coordination of ruthenium complexes may distort and even unwind the G-quadruplex so that further coordination of ruthenium to the linear DNA fragment occurred.     

The value of assessing cell proliferation in breast cancer

Using three methods to measure cell proliferation, namely DNA cell cycle; anti-proliferating cell monoclonal antibody (MAb) (Ki67, P145) analysis by flow cytometry; and the histological silver (argyrophilic) staining technique to visualize nuclear-organizing regions (AgNOR), twenty-two paired samples of primary breast tumour and axillary lymph node were analysed. The results showed variable levels of correlation between the methods used for the tumour group (r = 0.915, P <0.001 for Ki677 versus P145; r = 0.42, P <0.005 for percentage S/G2/M-phase versus P145; r = 0.16, P <0.5 for percentage S/G2/M-phase versus AgNOR; r = 0.400, P < 0.1 for Ki67 versus AgNOR). The lymph-node group showed slightly poorer correlations, yet involved nodes showed a consistently higher level of proliferation than non-involved nodes by all methods used. Overall, MAb binding of Ki67 or P145 was seen to be a good indicator of cycling cells, detecting G1-phase cells in addition to S/G2/M-phase cells indentified by the other methods used. However, no advantage was found over the usual DNA flow cytometric analysis of cells, which had clear prognostic value. AgNOR scores were found to be able to discriminate between diploid and aneuploid; and dividing and non-dividing cells, but areas of score overlap limited the application of this technique to that of a positive discriminator only.     

Re-evaluation of nestin as a marker of oligodendrocyte lineage cells

Maturation of oligodendrocyte progenitors (O2A) is characterized by morphological changes and the sequential expression of specific antigens leading to the formation of myelin membrane. Monoclonal antibodies A2B5, A007, anti-vimentin, and anti-galactocerebroside, recognize oligodendroglia at different stages of development. The neuroepithelial precursor marker nestin is also expressed by the oligodendroglial lineage; we have used enriched populations of progenitors isolated from neonatal rat brain cultures to further examine the cellular distribution of this intermediate filament protein. The phenotypic distribution of nestin positive cells among the oligodendrocyte lineage showed that 65% reacted with A2B5, whereas only 5% were A007(+), and 4% galactocerebroside(+). The remaining 25% of the cells were not labeled and had small cellular bodies devoid of processes, characteristic of the pre-O2A progenitor. Further analysis of the nestin(+) population showed that the majority of the cells were also vimentin(+). Antibody-dependent complement mediated cytolysis of A2B5(+) (O2A cells) and galactocerebroside(+) (mature oligodendrocytes) cells left a population of nestin(+) cells that were induced to proliferate in the presence of growth factors and to differentiate into A2B5(+) and galactocerebroside(+) cells. Proliferating cells maintained in the presence of platelet-derived growth factor or basic fibroblast growth factor retained nestin expression along with A2B5. By contrast, in serum-free medium nestin expression decreased while postmitotic cells acquired A007 and galactocerebroside. Our results suggest that nestin expression is a marker of pre-O2A cells that is maintained in proliferating glial progenitors, but is quickly down-regulated in postmitotic oligodendrocytes (A007(+)/galacto-cerebroside(+)) along with A2B5 and vimentin. However, other glial cells including type 2 astrocytes and some amoeboid microglia also share nestin expression.     

Procedure of RNA extraction which does not extract DNA and proteins from nervous tissue cells.

Several schemes of acidic extraction of RNA taken from biochemical or cytochemical literature were compared by means of ultraviolet and visible cytospectrophotometry. Basing on the data obtained an extraction scheme has been recommended which consists in the treatment of nervous tissue sections with 16 per cent perchloric acid at 4 degrees C for 48 h. This extraction has been shown to extract completely cellular RNA without extracting DNA and proteins from the nervous tissue cells. Using this scheme, ultraviolet cytospectrophotometry of neurons and glial cells was performed twice, before and after the selective RNA extraction, which enabled a calculation of RNA concentration by the difference of the absorption values. In mice, after 3 h-swimming, and in cats, after 2-week daily injections of adrenaline, a number of quantitative and qualitative RNA changes were found out in nerve and glial cell of mouse spinal cord and cat sympathetic ganglion with the aid of the recommended scheme of RNA extraction.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Insight into 5-aminolevulinic acid-induced modulation of cellular antioxidant metabolism to confer salinity and drought tolerance in maize

The current study investigated the comparative oxidative damage in two maize seedlings induced by saline, drought, and combined stress and the ameliorative role of two different doses (20 and 80 µM) of 5-aminolevulinic acid (ALA) against the above-mentioned stresses. Hydroponically grown 10-day-old maize (Zea mays, var. BARI Hybrid Maize-7 (BHM-7) and BARI Hybrid Maize-9 (BHM-9)) seedlings were exposed to 12 dS/m of saline solution, 200 mM mannitol-induced drought stress alone and their combined stress for 7 days. Result revealed that individual stresses retard the plant growth to some degrees; however, their combined stress has more detrimental effects, which might be correlated with lipid peroxidation (MDA)-induced oxidative stress in seedlings, enhanced Na⁺ /K⁺ ratio, and augmented generation of superoxide (O₂^•− ) and hydrogen peroxide (H₂O₂). In contrast, exogenous ALA supplementation at 20 µM concentration markedly recovered from chlorosis and growth inhibition, substantially scavenged reactive oxygen species (ROS) and MDA by preserving ionhomeostasis and relaxing oxidative stress; also, by boosting catalase (CAT) and glutathione S-transferase (GST), and exclusively via depressing the activity of lipoxygenase (LOX) antioxidant enzyme. On the contrary, 80 µM ALA made things worse; nevertheless, higher activities shown by other antioxidant enzymes; like, superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and glutathione peroxidase (GPX), which were related to lessen the oxidative damage by highly produced O₂^•− and H₂O₂ under combined stress. Non-denaturing gel electrophoresis was done for further confirmation. However, ALA importantly increased the photosynthetic pigment contents in both genotypes irrespective of doses. Nevertheless, GST might have assisted the plants to escape from the herbicidal effect by detoxification. However, in the combined stress condition, high ALA concentration may have some positive role to play. Our findings also showed that BHM-9 performed better than BHM-7. Therefore, ALA at lower concentration was effective for single stress of saline and drought, while higher concentration can improve plant survival under combined stress.