Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

Bacterial laccases: promising biological green tools for industrial applications.

Multicopper oxidases (MCOs) are a pervasive family of enzymes that oxidize a wide range of phenolic and nonphenolic aromatic substrates, concomitantly with the reduction of dioxygen to water. MCOs are usually divided into two functional classes: metalloxidases and laccases. Given their broad substrate specificity and eco-friendliness (molecular oxygen from air as is used as the final electron acceptor and they only release water as byproduct), laccases are regarded as promising biological green tools for an array of applications. Among these laccases, those of bacterial origin have attracted research attention because of their notable advantages, including broad substrate spectrum, wide pH range, high thermostability, and tolerance to alkaline environments. This review aims to summarize the significant research efforts on the properties, mechanisms and structures, laccase-mediator systems, genetic engineering, immobilization, and biotechnological applications of the bacteria-source laccases and laccase-like enzymes, which principally include Bacillus laccases, actinomycetic laccases and some other species of bacterial laccases. In addition, these enzymes may offer tremendous potential for environmental and industrial applications.

Improving the catalytic efficiency of Bacillus pumilus CotA-laccase by site-directed mutagenesis.

Bacterial laccases are potential enzymes for biotechnological applications because of their remarkable advantages, such as broad substrate spectrum, various reactions, high thermostability, wide pH range, and resistance to strongly alkaline environments. However, the use of bacterial laccases for industrialized applications is limited because of their low expression level and catalytic efficiency. In this study, CotA, a bacterial laccase from Bacillus pumilus, was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability. L386W/G417L, a CotA double-mutant, was constructed through site-directed mutagenesis. The catalytic efficiency of L386W/G417L was 4.3 fold higher than that of wild-type CotA-laccase, but the thermostability of the former was decreased than that of the latter and other mutants. The half-life (t 1/2) of wild-type and G417L were 1.14 and 1.47 h, but the half-life of L386W/G417L was only 0.37 h when incubating the enzyme at 80 °C. Considering the high catalytic efficiency of L386W/G417L, we constructed L386W/G417L/G57F, another mutant, to improve thermostability. Results showed that the half-life of L386W/G417L/G57F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity, but the residual activity of L386W/G417L was less than 40% when incubating the enzyme at 90 °C for 5 min. L386W/G417L was more efficient in decolorizing various industrial dyes at pH 10 than other mutants. L386W/G417L/G57F also exhibited an efficient decolorization ability. L386W/G417L/G57F is appropriate for biotechnological applications because of its high activity and thermostability in decolorizing industrial dyes. CotA-laccase may be further subjected to molecular modification and be used as an enhancer to improve decolorization efficiency for the physical and chemical treatment of dye wastewater.

Establishment of a markerless multiple-gene deletion method based on Cre/loxP mutant system for Bacillus pumilus.

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

Production of spore laccase from Bacillus pumilus W3 and its application in dye decolorization after immobilization.

Given that spore laccase from the Bacillus genus is heat- and alkali-resistant, it is more suitable for industrial applications than fungal laccase. To determine the optimal culture conditions for spore laccase production, the effects of Cu2+ concentration, oxygen content, and culture time on spore laccase production from Bacillus pumilus W3 were investigated. The optimal production parameters were 0.2 mM of Cu2+, 200 rpm shaking speed, 100 mL liquid loading, and 5 days of cultivation. Spore laccase was efficiently immobilized on amino-functionalized celite. When used in dye decolorization, the immobilized spore laccase removed 84.15% of methyl green and 69.70% of acid red 1 after 48 h of treatment. Moreover, the immobilized spore laccase retained 87.04% of its initial decolorization activity after six cycles in the decolorization of acid red 1. These insights into the culture conditions and immobilization of spore laccases should be useful in the development of spore laccase as a biocatalyst in the treatment of textile wastewater.

Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

Improving the decolorization activity of Bacillus pumilus W3 CotA-laccase to Congo Red by rational modification.

Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.

Molecular identification of the gene encoding porcine tristetraprolin (TTP).

Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element (ARE) binding sites. In this study, a novel porcine cDNA has been isolated by expressed sequence tag assembly and subsequently confirmed by RT-PCR analysis, and designated porcine TTP (poTTP). The open reading frame of the poTTP cDNA is 981 bp, encoding 326 amino acids. The poTTP gene is approximately 2.5 kb in size and contains a single intron. Southern blotting analysis demonstrated that it is a single copy gene. Real-time quantitative PCR analysis revealed that the poTTP gene is constitutively expressed in all detected tissues, and with the highest mRNA level in lymphoid tissues spleen and thymus. Recombinant His(6)-tagged poTTP protein and its two zinc finger mutants (C146G and H127I) were efficiently expressed and purified from Escherichia coli BL21 (DE3), respectively. In vitro, RNA-electrophoretic mobility shift assay confirmed a direct interaction between poTTP protein and porcine TNF-α (poTNF-α) mRNA ARE probe; this interaction was eliminated when using either two zinc finger mutants of poTTP. Consistently, mutations within the ARE region prevented the binding interaction between recombinant poTTP protein and poTNF-α mRNA ARE probe. These results indicate that poTTP is an ARE-binding protein that might regulate the turnover of certain mRNAs in vivo.

Gut microbiota of red swamp crayfish Procambarus clarkii in integrated crayfish-rice cultivation model.

Increasing evidences suggest that intestinal microbiota balance closely correlated with host's health status could affected by external environment. Integrated crayfish-rice cultivation model is a highly efficient artificial ecosystem widely practiced in subtropical China. Less information is available to estimate the influence response to the micro-ecology of crayfish intestine and so as to influence the biological processes. Thus, 16S rRNA high-throughput sequencing approach was employed to investigate the composition diversity and functions of bacterial community in the intestines of Procambarus clarkii farmed within this model. Results exhibited the highly diversity of microflora with dominant phyla Actinobacteria, Proteobacteria, Tenericutes, Firmicutes and Bacteroidetes. The genera of Candidatus Bacilloplasma and Ornithinibacter were presented as predominant population much exceeds in richness comparing to that of other genus. Despite the highly diversity in the bacterial community, the predicted functions indicated relative consistent in biological processing pathway. Collectively, significant richness of genes was observed involved in amino acid and carbohydrate metabolism and membrane transport processing. This study would contribute to the understanding of the impact of growth conditions on host-microbiota relation especially in aquatic animals.

Recombinant Horseradish Peroxidase C1A Immobilized on Hydrogel Matrix for Dye Decolorization and Its Mechanism on Acid Blue 129 Decolorization.

In this study, horseradish peroxidase C1A (HRP C1A) from Armoracia rusticana was expressed in Escherichia coli as an inclusion body. Subsequently, an active recombinant HRP C1A was obtained by refolding gradually using dilution-ultrafiltration. The recombinant HRP C1A was immobilized on agarose-chitosan hydrogel at 86.9 ± 2.5% of immobilization efficiency. After immobilization of the recombinant HRP C1A, the pH and temperature stability were improved and the reusability of the recombinant HPR C1A was achieved. The immobilized HRP C1A activity was retained above 80% after 6 cycles. The immobilized recombinant HRP C1A was used for the decolorization of four various dyes, including acid blue 129 (AB129), methyl blue (MB), methyl red (MR), and trypan blue (TB). The decolorization rates are all more than 70%, among which the decolorization effect of AB129 was the most significant (the decolorization rate was 76.3 ± 1.6%). Furthermore, a plausible decolorization pathway for AB129 was proposed based on the identified intermediates by ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS). This is the first report of the putative mechanism on the decolorization of AB129 by HRP.

Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis.

CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7-11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis.

Molecular cloning, genomic organization and expression analysis of the gene encoding bovine (Bos taurus) B-cell activating factor belonging to the TNF family (BAFF).

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine B-cell activating factor belonging to TNF family (bBAFF). The open reading frame (ORF) of this cDNA covers 843 bp, encoding 280 amino acids. The functional soluble part of bBAFF (bsBAFF) shows 96% and 91% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bBAFF genomic sequence consists of six exons and five introns, is approximately 30 kb in size, and maps to bovine chromosome 12q. Southern blotting analysis indicated that the bBAFF gene is a single copy gene. Real-time quantitative PCR (qPCR) analysis revealed that bBAFF is predominantly expressed in bovine lymphoid tissues PBLs and spleen. The predicted three dimensional (3D) structure of the bsBAFF monomer analyzed by "comparative protein modeling" revealed that it is very similar to its human counterpart. In western blotting analysis, His6-tagged bsBAFF protein expressed in E. coli could be recognized not only by an anti-His6.tag mAb but also by an anti-human sBAFF mAb, indicating immunological cross-reactivity occurs between bovine and human sBAFF protein.

Molecular characterization of cytokine TWEAK and its receptor Fn14 in pig (Sus scrofa).

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily (TNFSF). The interaction of TWEAK with its receptor fibroblast growth factor-inducible 14 (Fn14) regulates multiple cellular responses, including stimulation of proliferation, migration, apoptosis, angiogenesis, and induction of proinflammatory cytokines. This paper reports for the first time the molecular cloning of porcine TWEAK and Fn14 by EST and RACE strategies. The full-length cDNA of porcine TWEAK is 1327bp, including an open reading frame (ORF) of 747bp. Its genomic DNA consists of seven exons and six introns and is approximately 10kb in size by computer-assisted analysis. Sequence similarity at the amino acid level between porcine TWEAK and human or mouse was 95 and 92%, respectively. The full-length cDNA of porcine Fn14 contains 691bp, of which 390bp are the ORF. Sequence similarity at the amino acid level between porcine Fn14 and human, or mouse, or frog was 95, 93 and 64%, respectively. Real-time quantitative PCR (Q-PCR) analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in pig. Our results suggest that the TWEAK-Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families.

cDNA cloning, genomic structure, expression analysis and biological activity of porcine A Proliferation-Inducing Ligand (APRIL).

A Proliferation-Inducing Ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily. In this study, a novel cDNA has been isolated from pig spleen by homology cloning and 3'- and 5'-rapid amplification of cDNA ends (RACE) strategies and designates porcine APRIL (pAPRIL). The open reading frame (ORF) of this cDNA covers 756 bases, encoding 251 amino acids. The soluble part of pAPRIL shows 89% identity with its human counterpart at the level of the primary protein structure. The pAPRIL gene is approximately 2.1kb in size and comprises six exons and five introns. Southern blotting analysis indicated that the pAPRIL gene is a single copy gene. Real-time PCR analysis revealed that pAPRIL is constitutively expressed in various tissues. Recombinant His(6)-tagged psAPRIL protein was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. In vitro, purified recombinant psAPRIL protein co-stimulated the proliferation of porcine splenic B-cells in response to formalin-fixed Staphylococcus aureus Cowan 1 (SAC).

Cloning, expression and bioactivity of duck BAFF.

B cell activating factor (BAFF) belonging to the TNF family is critical for B cell survival and maturation. In the present study, we identified a duck BAFF cDNA, named dBAFF, by RT-PCR and RACE strategies. The open reading frame (ORF) of this cDNA encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like chicken BAFF (cBAFF), human BAFF (hBAFF) and mouse BAFF (mBAFF). The amino acid identity between biologically soluble dBAFF and cBAFF, hBAFF or mBAFF is 97, 78 and 71%, respectively. RT-PCR analysis showed the dBAFF gene is strongly expressed in the bursa of fabricius. Recombinant soluble dBAFF (dsBAFF) fused with NusA.tag was efficiently produced in Origami B (DE3) pLysS expression host strain. In vitro, purified dsBAFF was not only able to promote survival of bursa B cells, but also able to co-stimulate proliferation of mammalian B cells with anti-IgM. Furthermore, recombinant hsBAFF has a positive effect on duck bursa B cells survival. These findings indicate dBAFF plays an important role in survival and proliferation of duck B cells and because of its high conservation in the evolution, functional cross-reactivity exists between mammalian and duck BAFF.

Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis.

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.

cDNA cloning, expression and bioactivity of porcine BAFF.

B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805bp with a 702bp open reading frame, encoding 233 amino acids. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site corresponding to other identified BAFF homologues. The amino acid similarity between the functional soluble parts of pBAFF and human BAFF (hBAFF) or chicken BAFF (cBAFF) is 93% and 85%, respectively, with identity at the amino acid level was 88% and 76%, respectively. The characteristic of the three-cysteine residues of BAFF is conserved in pBAFF. RT-PCR showed that BAFF is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, kidney, thymus and PBLs. Recombinant soluble pBAFF (psBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. In vitro, purified psBAFF co-stimulates the proliferation of not only porcine B cells but also human B cells. In addition, hsBAFF binds to porcine B cells and has a positive effect on their proliferation. These findings indicate pBAFF plays an important role in proliferation of porcine B cells and functional cross-reactivity occurs between porcine and human BAFF. In vitro expression of bioactive psBAFF provides the basis for further investigation of its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic in pig. It also provides the basis for investigations on the role of BAFF in this important domestic species and an animal model for human diseases.

Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor.

B-cell activating factor (BAFF), belonging to the TNF family, is critical for B cell survival and maturation. cDNA of goose BAFF (gBAFF) was amplified from goose spleen by RT-PCR. The open reading frame (ORF) of gBAFF encodes a protein of 288-amino acid. The gBAFF shows 98, 92, 44 and 55% amino acid sequence identity with duck (dBAFF), chicken (cBAFF), mouse (mBAFF) and human BAFF (hBAFF), respectively. RT-PCR results showed that gBAFF mRNA is expressed in thymus and more highly expressed in the bursa of Fabricius and spleen. Recombinant soluble gBAFF (gsBAFF) expressed in Escherichia coli has molecular weight of approximately 19kDa. In vitro, purified gsBAFF was able to promote bursa B cells survival/proliferation in goose, duck and chicken. Furthermore, recombinant dsBAFF and csBAFF have a positive effect on goose, duck and chicken bursa B cells survival/proliferation. These findings indicate that gBAFF plays an important role in the survival/proliferation of goose B cells and, owing to its high evolutionary conservation, functional cross-reactivity exists between chicken, duck and goose BAFF.

Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria.

Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.