Microbial Metabolite Human Endogenous Metabolite

The secondary fermentation stage is critical for stabilizing composting products and producing various secondary metabolites. However, the low metabolic rate of mesophilic bacteria is regarded as the rate-limiting stage in composting process. In present study, two 3-Indoleacetic acid (IAA)-producing bacteria (Bacillus safensis 33C and Corynebacterium stationis subsp. safensis 29B) were inoculated to strengthen the secondary fermentation stage to improve the plant-growth promoting potential of composting products. The results showed that the addition of IAA-producing bacteria promoted the assimilation of soluble salt, the condensation and aromatization of humus, and the accumulation of dissolved organic nitrogen (DON) and dissolved organic carbon (DOC). The bioaugmentation strategy also enabled faster microbial community succession during the medium-late phase of secondary fermentation. However, the colonization of Bacillus and Corynebacterium could not explain the disproportionate increase of IAA yield, which reached up to 5.6 times compared to the control group. Deeper analysis combined with physicochemical properties and microbial community structure suggested that IAA-producing bacteria might induce the increase of salinity, which enriched halotolerant bacteria capable of producing IAA, such as Halomonas, Brachybacterium and Flavobacterium. In addition, the results also proved that it was necessary to shorten secondary fermentation time to avoid IAA degradation without affecting composting maturity. In summary, enhancing secondary fermentation of composting via adding proper IAA-producing bacteria is an efficient strategy for upgrading the quality of organic fertilizer.[1]

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Cytoplasmic male sterility (CMS) provides an irreplaceable strategy for commercial exploitation of heterosis and producing high-yielding hybrid rice. The exogenous application of plant growth regulators could improve outcrossing rates of the CMS lines by affecting floral traits and accordingly increase hybrid rice seed production. The present study aimed at exploring the impact of growth regulators such as gibberellic acid (GA3), 3-Indoleacetic acid(IAA), and naphthalene acetic acid (NAA) on promoting floral traits and outcrossing rates in diverse rice CMS lines and improving hybrid rice seed production. The impact of foliar applications of growth regulators comprising GA3 at 300 g/ha or GA3 at 150 g/ha + IAA at 50 g/ha + NAA at 200 g/ha versus untreated control was investigated on floral, growth, and yield traits of five diverse CMS lines. The exogenously sprayed growth regulators, in particular, the combination of GA3, IAA, and NAA (T3) boosted all studied floral, growth, and yield traits in all tested CMS lines. Moreover, the evaluated CMS lines exhibited significant differences in all measured floral traits. L2, L3, and L1 displayed the uppermost spikelet opening angle, duration of spikelet opening, total stigma length, xstyle length, stigma brush, and stigma width. In addition, these CMS lines exhibited the highest plant growth and yield traits, particularly under T3. Consequently, exogenous application of GA3, IAA, and NAA could be exploited to improve the floral, growth, and yield traits of promising CMS lines such as L2, L3, and L1, hence increasing outcrossing rates and hybrid rice seed production.[2]

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3-Indoleacetic acid (IAA) is a plant growth regulator that plays an important role in plant growth and development, and participates in the regulation of abiotic stress. To explore the effect of IAA on cadmium toxicity in Cinnamomum camphora, an indoor potted experiment was conducted with one-year-old C. camphora seedlings. The influence of IAA on cadmium accumulation, net photosynthetic rates, respiration, photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids), osmoregulatory substances (proline, soluble sugar and soluble protein) and the malondialdehyde content in C. camphora leaves treated with 30 mg kg-1 cadmium was analysed with or without the addition of 10 mg kg-1 IAA. Cadmium accumulation in the leaves of C. camphora with the addition of exogenous IAA was significantly higher than accumulation during cadmium stress without additional IAA (ca 69.10% after 60 days' incubation). During the culture period, the net photosynthetic rate in C. camphora leaves subjected to cadmium stress without the addition of IAA was up to 24.31% lower than that of control plants. The net photosynthetic rate in C. camphora leaves subjected to cadmium stress and addition of IAA was up to 30.31% higher than that of leaves subjected to cadmium stress without the addition of IAA. Chlorophyll a, total chlorophyll and carotenoid contents in the cadmium-stressed leaves without the addition of IAA were lower than those in the control treatment. The presence of IAA increased the chlorophyll a, total chlorophyll and carotenoid contents relative to the cadmium stress without the addition of IAA. The respiration rate and concentrations of proline, soluble sugar, soluble protein and malondialdehyde in C. camphora leaves subjected to cadmium stress without the addition of IAA were higher than those in the control. The addition of IAA reduced the respiration rate, and the concentrations of proline, soluble sugar, soluble protein and malondialdehyde in C. camphora leaves when compared with the cadmium stress without the addition of IAA. These results indicate that exogenous IAA improves photosynthetic performance and the growth environment of C. camphora by enhancing the net photosynthetic rate, increasing concentrations of osmoregulatory substances, removing reactive oxygen radicals and eliminating potential damage, thereby reducing the toxic effects of cadmium on C. camphora[3].

The current study purposed to investigate the 3-Indoleacetic acid (IAA) possible adverse impacts on hematological parameters, hepatorenal function, cardiac, and skeletal muscles as well as testes of rats and histopathological alterations of respective organs and to determine the extent of reversing any adverse impacts occurred in animals after IAA withdrawal. Rats were exposed orally to 500 mg/kg BW by gastric intubation once daily for 14 days, after which one-half was sacrificed and the remaining half left for a further 14 days without IAA exposure. The exposure of rats to IAA produced anemia, leukopenia, neutrophilia, lymphopenia, and a significant increase in activities of serum transaminase, gamma-glutamyl transferase, creatine kinase-myocardial band, creatine kinase-muscle type, and levels of serum creatinine, sodium, chloride, and potassium. Furthermore, serum levels of testosterone, gonadotropins, and leptin significantly declined. The changes in most of measured parameters continued after IAA withdrawal. Histopathological alterations in different tissues supported these changes. In conclusion, subacute exposure to IAA at a high concentration could exert hematotoxicity and toxic effects on many soft organs and its withdrawal led to incomplete recovery of animals. Thus, IAA should be used cautiously as extensive use of it at high concentrations can cause harmful effects on the environment, animals and human beings [4].

Three treatments were performed in this study, including uncontaminated soil + C. camphora (control), cadmium-contaminated soil (30 mg kg−1) + C. camphora (T1) and cadmium-contaminated soil (30 mg kg−1) + C. camphora + IAA (10 mg kg−1) (T2). Five replicates were used for each treatment. The concentration of cadmium in the soil was set in accordance with the “Soil environmental quality—Risk control standard for soil contamination of development land (GB3660–2018)” issued jointly by the Ministry of Ecological Environment and the State Administration for Market Regulation of the People's Republic of China. Solutions of 2 g L−1 CdCl2·2.5H2O (calculated from the Cd2+ concentration) and 0.5 g L−1 3-Indoleacetic acid/IAA solution were prepared. The final concentrations of cadmium and IAA in soil were 30 mg kg−1 and 10 mg kg−1 respectively and the seedlings were planted when the concentrations of Cadmium and IAA reached 30 mg kg−1 and 10 mg kg−1 respectively. Nitrogen (300 kg ha−1) and phosphorus (150 kg ha−1) were routinely applied as water-soluble fertilisers and these were diluted and applied as base fertilisers. Potted plants were placed on a light test table; each pot was 15 cm (diameter) × 24 cm (height) and contained 2.5 kg of soil. The locations of the pots were changed weekly. The pots were weighed and deionised water was added daily to keep the soil moisture at 50% of the saturated water content. During the culture period, light (light intensity = 8000–10 000 Lux) and dark periods were set at 12 h each. Plants were sampled on days 0, 15, 30 and 60, and physiological and biochemical indices such as net photosynthetic rates, photosynthetic pigments and contents of osmoregulators and MDA were measured [3].

Experimental procedure [4]
Thirty-six rats were divided randomly into three groups, with 12 rats in each group as follows:
Group I (control): rats were given only standard feed and water.
Group II (vehicle): rats orally received 0.5 ml olive oil orally by gastric intubation once daily for 14 days.
Group III (IAA): rats orally received IAA/3-Indoleacetic acid powder suspended in olive oil at concentration of 500 mg/kg BW by gastric intubation once daily for 14 days. The dose of IAA was selected based on the previously published studies (Furukawa et al. 2004). Bearing that in mind, the median lethal dose (LD50) of IAA in rats (oral treatment) is more than 500 mg/kg BW (Paley 2021). After 14 days of IAA exposure, the rats were maintained for another 14 consecutive days without any treatment.

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Sampling [4]
Sample collection during the experiment course occurred twice, after 14 days from exposure to 3-Indoleacetic acid/IAA and after 14 days of stopping exposure to IAA from the different experimental groups. Blood samples were collected from overnight fasted rats after being anesthetized by sodium pentobarbital by puncturing the retro-orbital venous sinus. The first part of blood specimens (1 ml) was collected in clean Wasserman tubes containing dipotassium salt of ethylenediamine tetraacetic acid (EDTA) for performing various hematological tests. The second part of blood specimens (1.5 ml) was collected in ordinary tubes and left to coagulate for centrifugation and separation of serum for performing the different biochemistry and hormonal assays. Rats were euthanized by decapitation after being anesthetized and the liver, kidneys, heart, skeletal muscle (extensor digitorum longus), and testes were excised quickly for histopathological studies.

Metabolism / Metabolites
Known metabolites of indole-3-acetic acid include indole-3-acetic acid-O-glucuronide. Indoleacetic acid (IAA) is a breakdown product of tryptophan metabolism and is typically produced by bacteria in the mammalian gut. Some endogenous IAA is also produced in mammalian tissues. It may be generated by the decarboxylation of tryptophan or the oxidative deamination of tryptophan.

Toxicity Overview
Uremic toxins, such as indole-3-acetic acid, can be actively transported to the kidneys via organic ion transporters, particularly OAT3. Elevated uremic toxin levels can stimulate the production of reactive oxygen species (ROS). This appears to be mediated by the direct binding of uremic toxins to or inhibition of NADPH oxidases, particularly NOX4, which is abundant in the kidneys and heart (A7868). ROS can induce a variety of different DNA methyltransferases (DNMTs) involved in the silencing of the KLOTHO protein. KLOTHO has been shown to play an important role in anti-aging, mineral metabolism, and vitamin D metabolism. Multiple studies have shown that in acute or chronic kidney disease, KLOTHO mRNA and protein levels are decreased due to elevated local ROS levels (A7869).

[1]. Inoculating indoleacetic acid bacteria promotes the enrichment of halotolerant bacteria during secondary fermentation of composting. J Environ Manage. 2022 Nov 15;322:116021.
[2]. Growth Regulators Improve Outcrossing Rate of Diverse Rice Cytoplasmic Male Sterile Lines through Affecting Floral Traits. Plants (Basel). 2022 May 12;11(10):1291.
[3]. Effects of exogenous 3-indoleacetic acid and cadmium stress on the physiological and biochemical characteristics of Cinnamomum camphora. Ecotoxicol Environ Saf. 2020 Mar 15;191:109998.
[4]. Assessment toxic effects of exposure to 3-indoleacetic acid via hemato-biochemical, hormonal, and histopathological screening in rats. Environ Sci Pollut Res Int. 2022 Dec;29(60):90703-90718.

Sodium indole-3-acetate is a member of the indole-3-acetic acid family. Indole-3-acetic acid is a monocarboxylic acid formed by replacing one methyl hydrogen atom in the acetic acid molecule with a 1H-indole-3- group. It is a plant hormone, a human metabolite, a plant metabolite, a mouse metabolite, and an auxin. It is a monocarboxylic acid and a member of the indole-3-acetic acid family. It is the conjugate acid of indole-3-acetic acid esters. Indoleacetic acid is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). Indole-3-acetic acid has also been reported in hops, plants of the Barancia genus, and other organisms with relevant data. Indoleacetic acid is a uremic toxin. Based on chemical and physical properties, uremic toxins can be divided into three main categories: 1) small, water-soluble, non-protein-bound compounds, such as urea; 2) small, lipid-soluble and/or protein-bound compounds, such as phenols; 3) larger so-called medium-molecular-weight compounds, such as β2-microglobulins. Long-term exposure to uremic toxins can lead to various diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. Indoleacetic acid (IAA) is a breakdown product of tryptophan metabolism and is normally produced by bacteria in the mammalian gut. IAA is also endogenously produced in mammalian tissues. IAA can be produced through tryptophan decarboxylation or tryptophan oxidative deamination. Indoleacetic acid (IAA) is usually present in low concentrations in urine, but its concentration has been found to be elevated in the urine of patients with phenylketonuria (PKU). In 1933, Kogl discovered this using substances extracted from human urine. Indoleacetic acid is also an important plant hormone. Specifically, IAA is a member of a family of plant hormones known as auxins. IAA is generally considered the most important natural auxin. Plant cells synthesize IAA using tryptophan. IAA and some of its derivatives can be oxidized by horseradish peroxidase (HRP) into cytotoxic substances. IAA only becomes toxic after oxidative decarboxylation; the effect of IAA/HRP is thought to be partly attributed to the formation of methylene indolone, which can bind to DNA bases and protein thiols. IAA/HRP can serve as a basis for targeted cancer therapy, including antibody, polymer, or gene-guided therapy, representing a potential new role for plant auxins in cancer treatment (A3268, A3269). Indole-3-acetic acid (IAA) is produced by Saccharomyces cerevisiae. IAA is a metabolite found or produced in cerevisiae. Inoculation with exogenous IAA-producing bacteria during the secondary fermentation of compost promotes the assimilation of soluble salts, condensation and aromatization of humic substances, and the accumulation of IAA, DON, and DOC. The microbial composition depends primarily on physicochemical properties and fermentation time, while inoculation with IAA-producing bacteria accelerates later-stage microbial succession. The large accumulation of IAA in the early stages of secondary fermentation may be closely related to the enrichment of salt-tolerant bacteria, which may be induced by the increased salinity caused by exogenous IAA-producing bacteria. Furthermore, the proliferation of various IAA-degrading bacteria in the later stages of composting indicates the need to shorten the secondary fermentation time to... This allows for the retention of more IAA without affecting compost maturity. These results provide new ideas for optimizing composting processes. Aerobic composting technology. [1]

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Compared with untreated plants, foliar spraying of the combination of gibberellin, indoleacetic acid, and naphthaleneacetic acid significantly improved flowering, growth, and yield traits in all evaluated CMS lines. In addition, the evaluated CMS lines showed different genetic behaviors in terms of flowering traits, plant growth, and yield. L2 and L1 showed the highest evaluated flowering traits, plant growth, and yield traits, especially under the T3 treatment. Therefore, foliar spraying of the combination of GA3, IAA, and NAA appears to be a way to improve the promising CMS lines L2 and L 1. An effective tool for cross-pollination to improve cross-pollination rate and hybrid seed yield. [2] Cadmium stress reduced the net photosynthetic rate and photosynthetic pigment content (chlorophyll a, total chlorophyll and carotenoids) of camphor tree leaves, increased the respiration rate, and increased the content of proline, soluble sugar, soluble protein and malondialdehyde (MDA). The content of these components decreased. The addition of exogenous indoleacetic acid (IAA) can increase the net photosynthetic rate of camphor tree leaves under cadmium stress, slow down respiration, reduce the content of proline and soluble sugar, alleviate the effects of cadmium stress, and promote the accumulation of cadmium in camphor trees. In summary, the application of IAA helps camphor trees adapt to cadmium stress and increase the accumulation of cadmium. Therefore, camphor trees have the function of remediating heavy metal pollution. Potential for soil contamination. [3]

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Considering the results of this study, I believe that subacute exposure to high concentrations of IAA can cause hematologic toxicity, manifested as decreased red blood cell count parameters, leukopenia, liver and kidney dysfunction, and various toxic effects on the heart, testes, and skeletal muscle. Changes in blood biochemistry and hormone test results, as well as histological structures of different organs, revealed these toxic effects. Furthermore, within the timeframe of the experimental study, the animals did not fully recover from the adverse effects after discontinuation of indoleacetic acid (IAA), and therefore may require a longer recovery period. Therefore, IAA should be used with caution, as large-scale use at high concentrations can have harmful effects on the environment, animals, and humans. [4]

C10H8NNAO2

197.17

197.045

6505-45-9

3-Indoleacetic acid;87-51-4

23677501

White to off-white solid powder

165-169ºC(lit.)

171ºC

0.46

1

2

2

14

210

0

[Na+].[O-]C(C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)=O

YGSPWCVTJRFZEL-UHFFFAOYSA-M

InChI=1S/C10H9NO2.Na/c12-10(13)5-7-6-11-9-4-2-1-3-8(7)9;/h1-4,6,11H,5H2,(H,12,13);/q;+1/p-1

sodium;2-(1H-indol-3-yl)acetate

6505-45-9; Indole-3-acetic acid sodium salt; DTXSID901036174; DTXCID801520384; 800-071-5; Sodium 2-(1H-indol-3-yl)acetate; 1H-Indole-3-acetic acid, monosodium salt; sodium;2-(1H-indol-3-yl)acetate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)