Azo dyes and human health: A review.

Synthetic azo dyes are widely used in industries. Gerhardt Domagk discovered that the antimicrobial effect of red azo dye Prontosil was caused by the reductively cleaved (azo reduction) product sulfanilamide. The significance of azo reduction is thus revealed. Azo reduction can be accomplished by human intestinal microflora, skin microflora, environmental microorganisms, to a lesser extent by human liver azoreductase, and by nonbiological means. Some azo dyes can be carcinogenic without being cleaved into aromatic amines. However, the carcinogenicity of many azo dyes is due to their cleaved product such as benzidine. Benzidine induces various human and animal tumors. Another azo dye component, p-phenylenediamine, is a contact allergen. Many azo dyes and their reductively cleaved products as well as chemically related aromatic amines are reported to affect human health, causing allergies and other human maladies.

Carcinogenicity, allergenicity, and lupus-inducibility of arylamines.

Arylamines are widely used in food, drugs, and cosmetics as well as other industries. These chemicals are present ubiquitously in cigarette smoke, smoke emitted from cooking fume hoods as well as are generated by diverse industries. Arylamines can be generated by cleavage of azo dyes by intestinal and skin microbiota. Some arylamines are used as drugs while others are constituents of human metabolism. Many of the arylamines are mutagenic and carcinogenic. They are generally recognized as the major cause of human bladder cancer, but arylamines can induce cancers of other organs in humans and animals. Some arylamines are allergenic, causing lupus like syndrome, or other maladies. In view of their unbiquitious nature and the diseases they cause, arylamines are probably the most important chemicals causing health problems.

Occurrence, uses, and carcinogenicity of arylamines.

Arylamines are chemically synthesized and contained in oxidants, epoxy polymers, explosives, fungicides, pesticides, colorants, polyurethanes, and used in rubber, pharmacology, cosmetics, and other chemical industries. Many arylamines are ubiquitously present in cigarette smoke, cooking fume hoods, foods, automobile exhaust, industrial sites, etc. Some arylamines can be generated through azo reduction by intestinal, skin, and environmental microorganisms from azo dyes that are widely used. Arylamines can also be generated by reduction of the nitro-group containing polyhydrated hydrocarbons including muntions. Some arylamines are released by burning nitrogen containing organic materials at high temperatures. Some medical drugs are also arylamines. Furthermore, many arylamines are essential constituents of normal metabolism or the result of abnormal metabolism or dietary sources. Some arylamines are mutagenic, carcinogenic or the cause of other kinds of maladies. Some arylamine are considered the major etiological agents of bladder tumors in humans and animals but may also induce other types of cancers in various organs. The organ, tissue, and species specificity of the arylamine-inducing carcinogenesis may be determined by their availability, distribution, and the presence of metabolic activation/detoxicification enzymes of each organ or tissue of different species. The ubiquitous arylamines, therefore, pose serious hazards to human health and environment. This article will address the occurrence, uses, carcinogenicity, and other arylamines-induced diseases.

Comparative genotoxicity of 3-hydroxyanthranilic acid and anthranilic acid in the presence of a metal cofactor Cu (II) in vitro.

Several clinical studies have reported that an increase in excretion of tryptophan metabolites 3-hydroxyanthranilic acid (3-OHAA), anthranilic acid (AA) and other metabolites in the urine of bladder cancer patients are implicated to play a role in the etiology of bladder cancer; however the mechanisms involved are unknown. The present study compares the genotoxicity of tryptophan metabolites AA and 3-OHAA to cause mutagenesis in vitro. The DNA damage effects of tryptophan metabolites were analyzed using plasmid relaxation assay performed with AA and 3-OHAA at various concentrations between 50µM and 400µM in the presence of plasmid DNA pSP-72. Both AA and 3-OHAA did not show any plasmid relaxation activity when tested alone. However, 3-OHAA in the presence of metal cofactor Cu (II) induced plasmid relaxation by causing nicks in the plasmid. This effect was not observed in the presence of other metal cofactors Fe (II) and Mn (III). Cu (II) at increasing concentrations between 5µM and 20µM and in the presence of 100µM 3-OHAA showed an apparent dose-response in causing DNA strand breaks. The Cu (II) mediated mutagenic activation of 3-OHAA was further investigated using Ames Salmonella/microsome mutagenicity assay with reactive oxygen species (ROS) sensitive tester strain Salmonella TA102. When 100µg of 3-OHAA per plate was incubated with Cu (II) a significant increase in TA102 revertants was observed with an increase in the concentration of Cu (II) from 2.5µg to 50µg. In contrast, AA with Cu (II) at such low concentration was unable to cause any significant increase in number of the TA102 revertants. This evidence for mutagenicity with only 3-OHAA and Cu (II) but not AA suggests the presence of hydroxyl group at ortho position to amino group in 3-OHAA structurally, is critical in reacting with Cu (II) to generate genotoxicity.

Anti-Cancer Effects of Protein Extracts from Calvatia lilacina, Pleurotus ostreatus and Volvariella volvacea.

Calvatia lilacina (CL), Pleurotus ostreatus (PO) and Volvariella volvacea (VV) are widely distributed worldwide and commonly eaten as mushrooms. In this study, cell viabilities were evaluated for a human colorectal adenocarcinoma cell line (SW480 cells) and a human monocytic leukemia cell line (THP-1 cells). Apoptotic mechanisms induced by the protein extracts of PO and VV were evaluated for SW480 cells. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of CL, PO or VV. Apoptosis analysis revealed that the percentage of SW480 cells in the SubG(1) phase (a marker of apoptosis) was increased upon PO and VV protein-extract treatments, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death. The PO and VV protein extracts induced reactive oxygen species (ROS) production, glutathione (GSH) depletion and mitochondrial transmembrane potential (ΔΨ(m)) loss in SW480 cells. Pretreatment with N-acetylcysteine, GSH or cyclosporine A partially prevented the apoptosis induced by PO protein extracts, but not that induced by VV extracts, in SW480 cells. The protein extracts of CL, PO and VV exhibited therapeutic efficacy against human colorectal adenocarcinoma cells and human monocytic leukemia cells. The PO protein extracts induced apoptosis in SW480 cells partially through ROS production, GSH depletion and mitochondrial dysfunction. Therefore, the protein extracts of these mushrooms could be considered an important source of new anti-cancer drugs.

Enhancement of temozolomide-induced apoptosis by valproic acid in human glioma cell lines through redox regulation.

Temozolomide (TMZ) is an oral alkylating agent that has been widely used in the treatment of refractory glioma, although inherent and acquired resistance to this drug is common. The clinical use of valproic acid (VPA) as an anticonvulsant and mood-stabilizing drug has been reported primarily for the treatment of epilepsy and bipolar disorder and less commonly for major depression. VPA is also used in the treatment of glioma-associated seizures with or without intracranial operation. In this study, we evaluated the potential synergistic effect of TMZ and VPA in human glioma cell lines. Compared with the use of TMZ or VPA alone, concurrent treatment with both drugs synergistically induced apoptosis in U87MG cells as evidenced by p53 and Bax expression, mitochondrial transmembrane potential loss, reactive oxygen species production, and glutathione depletion. This synergistic effect correlated with a decrease in nuclear translocation of the nuclear factor-erythroid 2 p45-related factor and corresponded with reduced heme oxygenase-1 and γ-glutamylcysteine synthetase expression. Pretreatment with N-acetylcysteine partially recovered the apoptotic effect of the TMZ/VPA combination treatment. The same degree of synergism is also seen in p53-mutant Hs683 cells, which indicates that p53 may not play a major role in the increased proapoptotic effect of the TMZ/VPA combination. In conclusion, VPA enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism. The TMZ/VPA combination may be effective for treating glioma cancer and may be a powerful agent against malignant glioma. This drug combination should be further explored in the clinical setting.

Possible roles of excess tryptophan metabolites in cancer.

Tryptophan is metabolized through serotonin, indole, and kynurenine (KN) pathways. Uptake of an excess amount of tryptophan accompanied with vitamin B6 deficiency may result in the accumulation of higher concentrations of metabolites mainly from the KN pathways in the bladder. These metabolites could interact with nitrite to become mutagenic nitrosamines. They could be a promoter in the initiator-promoter model of carcinogenesis. They produced bladder cancer when implanted in the bladder. They also interact with transition metals copper or iron to form reactive radicals or reactive oxygen species (ROS). Some metabolites, 3-hydroxy-anthranilic acid, were autooxidized to mutagenic cinnabarinic and anthranilyl radical intermediates. These radical intermediates could also be ligands that interact with aryl hydrocarbon receptor (AhR) and induce xenobiotic metabolizing enzymes (XMEs) to metabolize contaminated carcinogens. When tryptophan is exposed to either visible or UV light, a photoproduct of 6-formylindolo[3,2b]-carbazole is formed, which has a very high affinity for the AhR that plays a role in carcinogenesis. This review gives an insight into various mechanisms through which tryptophan metabolites cause carcinogenesis. It could be concluded that tryptophan metabolites play a complementary role in promoting carcinogenesis along with carcinogens like aflatoxin, CCl(4) , 2-acetylaminofluorene, 4-aminobiphenyl, 2-naphthylamine, or N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide. The underlying mechanisms could be their autoxidation, exposure to either visible or UV light, interaction with nitrite or transition metals to form reactive intermediates, serving as ligands to interact with an AhR that is known to play a role in carcinogenesis through induction of XMEs. Further research is warranted.Environ.

Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA.

Quercetin (QT) and Taxifolin (TF) are structurally similar plant polyphenols. Both have been reported to have therapeutic potential as anti-cancer drugs and antioxidants. Mutagenic effects of QT and TF were evaluated using Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA tester strains. Either in the presence or absence of S9 mix, QT was mutagenic to TA102 and WP2 uvrA. However, the mutagenicity of QT was significantly enhanced in the presence of S9 mix. Likewise, in the presence of Iron (Fe2+) and NADPH generating system (NGS) and absence of S9 mix, QT induced significantly high mutations in both TA102 and WP-2 uvrA. Mutagenicity of QT decreased in both strains in the presence of Iron (Fe2+) or NGS alone. TF was not mutagenic in the presence or absence of S9 mix in both TA102 and WP-2 uvrA 2, regardless of the presence of iron or NGS. Incorporation of antioxidants (ascorbate, superoxide dismutase (SOD), catalase (CAT)) and/or iron chelators (desferroxamine (DF) and ethylenediamine-tetraacetate (EDTA)) in the test systems markedly decreased QT-induced mutations in both tester strains. These results suggest that QT but not TF, could induce mutations in the presence or absence of rat liver S9 or Iron (Fe2+) and NGS in both tester strains by redox cycling and Fenton reactions to produce oxygen free radicals. Our results indicate that a minor structural variation between the two plant polyphenols could elicit a marked difference in their genotoxicities. These results provide a basis for further study into the potential use of QT in combination with iron supplements.

Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells.

This paper reports that a novel protein extract isolated from Calvatia lilacina (CL) can induce cell death against four types of human colorectal cancer cells. Importantly, CL was shown to be free of apoptotic effects against normal rat liver cells. We have also identified that CL-induced glutathione (GSH) depletion is the major contributor responsible for the apoptotic cell death induction of SW 480 cells, as evidenced by the observation that exogenously added N-acetylcysteine (NAC), or GSH, but not vitamin C, could offer a near complete protection of CL-treated cells against apoptotic cell death. Furthermore, the participation of reactive oxygen species (ROS) evoked a drop in the transmembrane potential (Delta Psi(m)) in the CL-induced apoptotic cell death. This observation can only be deemed as a minor pathway due to the fact that cyclosporine A (CyA) could only partially rescue the CL-treated cells from apoptotic cell death. Likewise, despite the fact that CL could induce the upregulation of Bax, its knockdown via siRNA (48 h) failed to completely mitigate apoptotic cell death, indicating that its role in this apoptotic process was insignificant. To further explore the possible underlying mechanism associated with CL-induced GSH depletion, we proceeded to determine the effect of CL on the cellular gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme responsible for GSH biosynthesis, and demonstrated that indeed gamma-GCS could be repressed by CL. Taken together, we report here for the first time that the anticancer effect of CL on human colorectal cancer cells is mediated through GSH depletion mechanism rather than a ROS-mediated killing process. This functional attribute of CL can thus provide the basis for the strategic design of a treatment of colorectal cancer.

Synthesis and biological evaluation of novel C(6) modified baicalein derivatives as antioxidative agents.

Baicalein, one of the major flavones, was found to be responsible for the antioxidative activity of the traditional Chinese medicinal herb Huang-Qin ( Scutellaria baicalensis Georgi), which is widely used as an antioxidative, anti-inflammatory, and antitumor agent. The hydroxyl group of the A ring of the baicalein was alkylated at position 6 with terpenoids such as prenyl, geranyl, and farnesyl groups, and their free radical scavenging activities and glutathione (GSH) depletion capacities were examined. Their free radical scavenging activity was measured according to the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(*+)) scavenging method. Baicalein and newly synthesized baicalein derivatives were found to be good free radical scavengers. Flow cytometrical method was employed to measure the intracellular antioxidative activity and GSH depletion capacity of these derivatives in human acute monocytic leukemia cell line (THP-1). It was also found that baicalein and its derivatives could decrease the levels of exogenous cumene hydroperoxide and H2O2 in THP-1 cells. These compounds also could significantly inhibit the intracellular GSH depletion induced by cumene hydroperoxide in THP-1 cells. The production of cumene hydroperoxide-induced Bax, a pro-apoptotic related protein, could also be inhibited by baicalein and its derivatives. These results suggested that baicalein and its derivatives could be beneficial to human health.

Surface-enhanced Raman spectroscopy using silver nanoparticles on a precoated microscope slide.

A chemical reduction method was applied to deposit nano silver particles on a frosted microscope slide, precoated with indium tin oxide. The substrate was used to collect the surface-enhanced Raman spectrum of N1'-ethyl indirubin monooxime (EIM), a potential chemical for pharmaceutical application. From the observed surface-enhanced Raman scattering (SERS) spectrum, EIM might interact with silver surface through the lone pair electrons of the oxime nitrogen atoms. Crystal violet (CV) and p-nitrobenzoic acid (PNBA) were also used to test the SERS probe capability of the substrate. The surface morphorlogy of the substrate has been characterized by using scanning electron microscope (SEM) and atomic force microscope (AFM). The elementary composition was identified with Edex and X-ray element scanning.

The metabolic activation of 2-aminofluorine, 4-aminobiphenyl, and benzidine by cytochrome P-450-107S1 of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa, namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa. We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5' and 3' untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYP107S1 knockout strains were selected on 1000 microg/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYP107S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYP107S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYP107S1 in P. aeruginosa.

Effects of various plant polyphenols on bladder carcinogen benzidine-induced mutagenicity.

Benzidine (Bz), a human bladder carcinogen, was strongly mutagenic to Salmonella TA102 tester strain in the Ames Salmonella microsome/mutagenicity assay in the presence of rat liver S9 mix. Various non-mutagenic plant polyphenols were included in the assay to test their inhibitory effects on the Bz-induced mutations. Coumestrol, ellagic acid (EA), (-)-epicatechin (EC), (-)-epichatechingallate (ECG), gallic acid (GA), (-)-gallocatechin (GC), plumbagin, propyl gallate (PG), taxifolin, and 2,2',4'-trihydroxychalcone were found to have a strong inhibitory effect on Bz-induced mutations. (-)-Epigallo-catechingallate (EGCG), fisetin, (-)-gallocatechingallate (GCG), and piceatannol were moderately inhibitory to the mutations; whereas, (-)-catechin, (-)-catechingallate (CG), and reseveratrol were weakly inhibitory to the mutations. (-)-Epigallocatechin (EGC) and 7,3',4'-trihydroxy isoflavon were not inhibitory to the Bz-induced mutations. Isoliquirtigenin, quercetin dihydrate, and rhein were found to be mutagenic in tester strain TA102. Benzidine mediated lipid peroxidation was conducted employing the thiobarbituric acid reactive substances (TBARS) assay using linoleic acid as a substrate. In the presence of rat liver S9 mix, Bz could cause lipid peroxidation as an outcome of production of oxygen free radicals. Incorporation of the above mentioned non-mutagenic plant polyphenols significantly inhibited benzidine mediated lipid peroxidation in a time dependent manner. These polyphenols also effectively reduced the iron mediated lipid peroxidation. Thus, it is concluded that the inhibition of oxidative mutagenicity of Bz by plant polyphenols could be due to an inhibitory effect of plant polyphenols on the bioactivating enzymes such as cytochrome P-450 and peroxidase and the chelation of iron present in the cytochrome P-450 in the S9 mix. Thus, these plant polyphenols play a significant inhibitory role on Bz-induced mutagenicity.

Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species.

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3-dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3,5,5-tetra-methylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N,N-diacetylbenzidine were not mutagenic. In addition, 3,3-dichlorobenzidine and 4,4-dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4-Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS.

p-Phenylenediamine induces p53-mediated apoptosis in Mardin-Darby canine kidney cells.

The mechanism of toxicity p-phenylenediamine (p-PD), a component of human permanent hair dye and a suspected carcinogen, on the growth of Mardin-Darby canine kidney cells (MDCK) was investigated. With the analysis of flow cytometry, a dose-dependent accumulation of the sub-G1 peak and the G0/G1-phase arrested in cell cycle, and time-dependent induction of apoptosis after staining with Annexin V-Fluorescein and propidium iodide were observed. After the treatment of cells with p-PD, dose dependent DNA fragmentation shown by gel electrophoresis, the reduction of membrane potential (DeltaPsim) by mitochondria membrane depolarization and the increase of the expression of p53 protein in cells, suggested that the effect of p-PD on overall viability and cell numbers is mediated by an increase in apoptosis.

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes.

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.

Metabolic activation of bladder procarcinogens, 2-aminofluorene, 4-aminobiphenyl, and benzidine by Pseudomonas aeruginosa and other human endogenous bacteria.

Pseudomonas aeruginosa, an opportunistic pathogen of the human urinary tract, and other selected human endogenous bacteria were investigated for metabolic activation of the bladder procarcinogens, 2-aminofluorene (2-AF), 4-aminobiphenyl (4-AB), and benzidine (Bz). The cell-free extracts of Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Staphylococcus saprophyticus, Klebsiella pneumoniae, and intestinal anaerobes, Bacteroides fragilis, Clostridium perfringens, and Eubacterium aerofaciens produced increased histidine revertant frequencies with the tester strain Salmonella typhimurium TA98 in the Ames Salmonella mutagenicity assay. In addition, the cell-free extracts of Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens each showed the presence of a cytochrome P450 absorption peak in the carbon monoxide (CO) difference spectrum. This was not demonstratable for the other bacteria. Our findings indicate that human endogenous bacteria, which are opportunistic pathogens of the urinary bladder, can metabolically activate the bladder procarcinogens 2-AF, 4-AB, and Bz into mutagens. The metabolic activation by Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens is mediated by a cytochrome P450 enzyme. For those organisms that induced metabolic activation but did not show a P450 absorption peak with the cell-free extracts, other oxidative enzymes may be involved.

Attachment and Proliferation of Bacteria on Meat.

The attachment of bacteria ( Serratia marcescens , Staphylococcus aureus , Streptococcus faecalis , Salmonella arizonae , Pseudomonas aeruginosa , and Listeria monocytogenes ), to lean muscle tissue and fat tissue was investigated. The number of cells attached to the meat was directly proportional to the initial cell concentrations present. There was no significant difference in the number of cells attached between the lean muscle tissue and fat tissues among the organisms tested. All bacteria tested except P. aeruginosa proliferated better on the lean muscle tissues than on the fat tissue at ambient temperature for 72 h. No significant attachment competition to tissue samples was seen between L. monocytogenes and P. aeruginosa , however, the numbers of P. aeruginosa were greater than L. monocytogenes (after 24 h). Similarly, no competitive attachments between S. aureus and S. marcescens , S. faecalis and S. arizonae were observed; but the numbers of S. marcescens were greater than S. aureus , and S. arizonae were greater than S. faecalis , when the inoculated meat was incubated at room temperature for 24 h.