Poultry Manure Induced DNA Damage


Redolent compounds are emitted from buildings, animal feed surfaces, manure storage and other treatment units, fodder piles and a variety of other emission sources [1]. Emission of redolent compounds from chicken sheds leads to an odour crisis in the contiguous area, resulting in complaints from residents or has some ill health effects [2]. An obnoxious odour is associated with poultry manure, which is a result of a combination of approximately 150 different compounds, including aldehydes, amines, ammonia, hydrogen sulphide, mercaptans, sulphur compounds and esters [2—4]. Odour is mainly due to the products formed upon the decomposition of chicken faeces, feathers and other litter by certain aerobic and anaerobic microorganisms. Each of these sources mentioned above has a distinct emission profile, which is unpredictable and remains irregular during the day and throughout the year [1].

Therefore, an effectual, helpful and efficient odour removal technique needs to be developed to tackle the great challenge of their disposal. Previous studies have demonstrated viable treatment of poultry manure with the mineral microbial preparation, which has successfully reduced odorants/redolents from the feedstock. These odorants are ammonia, dimethylamine (DMA), trimethylamine (TMA), isobutyric acid and other redolent compounds. A novel microbial mineral preparation composed of perlite and bentonite with spray-dried microorganisms is being used for the active removal of odours from poultry manure [5,6].

Ammonia, DMA, TMA, indole, phenol and butyric acid are the most general and regular compounds present in poultry manure [7-9]. Their health effects on humans and animals are well-known, and several studies have been done in-depth [10].

To summarise, these redolent compounds with extended exposure can have the following health effects:

  • • Pain in mucosal membranes in the respiratory tract
  • • Tracheal irritation
  • • Air sac inflammation
  • • Conjunctivitis
  • • Dyspnoea
  • • Respiratory tract damage
  • • Reddening
  • • Corneal clouding
  • • Reduction in respiratory rate
  • • Central nervous system disturbances [10]

There is still inadequate and paucity of in-vitro data linked with the cytotoxicity and genotoxicity of poultry redolent compounds pertaining to the application of cell lines. Therefore, this chapter deals with the cytotoxicity assay procedures and the associated requirements of the above-mentioned redolent compounds by employing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and PrestoBlue assays using chicken Leghorn Male Hepatoma (LMH) cell line [10]. These protocols will serve as appropriate for the concentrations of the redolent compounds regarding genotoxicity testing. Investigation for the mechanisms of toxicity of above-mentioned compounds redolent compounds can also be examined if cytotoxicity is an adjunct to genotoxicity, which can pilot to transformations and cancer.

The aim of the present chapter is to devise protocols for the measurement of genotoxicity (the measurement of DNA damage by employing the Comet assay) and cytotoxicity (necrosis in lactate dehydrogenase assay, along with IC5() values) of the above-mentioned redolent compounds from poultry manure in the chicken LMH cell line in vitro. In addition, the microscopic examination of any morphological changes in the cellular nuclei can also be studied by staining (apoptosis or necrosis after fluorescent staining).


Materials and Methods

Chemicals: Ammonia, DMA, TMA, indole, phenol, butyric acid, Waymouth’s medium with no supplements and sterilised filters (0.22 pM pore size filter, membrane solutions).

Note: All compounds need to be prepared freshly on the day of the experiment.

LMH Cell Culture

  • • The chicken liver hepatocellular carcinoma cell line, LMH, is to be used in the experiments as a model cell line.
  • • Target of redolent compounds is first on the respiratory tract, and from there they are transported with blood to other organs, and then to the liver, which then participates in their detoxification.
  • • The culturing of LMH cells were in the form of a monolayer as described earlier [10]. Shortly after that, it was again cultured in T75 flasks which were collagen-coated in a Waymouth’s medium with 7.5% sodium bicarbonate, 10% heat-inactivated foetal bovine serum (FBS), 25 mM HEPES and antibiotics (100 lU/mL penicillin and 100 pg/mL streptomycin).
  • • The cells were then incubated in a CO, incubator at 37°C in 5% CO, for 7 days to reach 80% confluence. These confluent cells were then detached with TrypLE™ Express for 10 min at 37°C, while remaining suspended in sterile PBS, aspirated off the plastic flask, which was then centrifuged (182 x g, 5 min), decanted and then re-suspended in fresh medium.
  • • These cells were then ready to use if a minimum of 90% viability was obtained when tested by Trypan blue exclusion.

Comet Assay (SCGE—Single Cell Gel Electrophoresis Assay) – Protocol


  • • The finishing concentration of LMH cells in every sample to be tested was adjusted to 105 cells/mL.
  • • The cells in non-supplemented Waymouth’s medium were then incubated with precise concentrations of each redolent compound at 37°C for 60 min.
  • • The absolute series of concentrations of the redolent compounds in culture were selected based on the available studies where IC50 values of these redolent compounds have been determined. According to literature, it is 0.001%-0.006% for DMA and ammonia, 0.001%-0.12% for TMA. 0.003%-0.5% for butyric acid and Indole and 0.0004%-0.1% for phenol [10].
  • • The IC5() values of the above-mentioned redolent compounds by MTT and PrestoBlue assays, which were 0.02%-0.08% for ammonia; 0.03%-0.06% for DMA; 0.02%-0.08% for TMA; 0.11%-0.32% for butyric acid; and 0.06% for indole, depending on the incubation time and the assay [10].
  • • The Comet assay was then performed under alkaline conditions (pH > 13), as per the procedure of Blasiak and Kowalik [11,12].
  • • In short, after their incubation, the cells were centrifuged (182 x g, for 15 min, at 4°C). They were then decanted and suspended in 0.75% low melting point Agarose, which was layered on to slides pre-coated with 0.5% normal melting point Agarose and lysed at 4°C for 60min in a buffer containing 2.5M NaCl, 1% Triton X-100, lOOmM EDTA and lOmM Tris, pH 10.
  • • These slides were then placed in an electrophoresis unit, and the DNA was allowed to unwind for 20min within an electrophoretic solution containing 300 mM NaOH and 1 mM EDTA.
  • • Electrophoresis was then conducted at 4°C for 20 min at an electric field strength of 0.73 V/cm (300 mA).
  • • Finally, the slides were neutralised in distilled water, and staining was performed with 2.5 pg/mL propidium iodide, and covered with coverslips. The slides were then observed at 200x magnification under a fluorescence microscope connected to a video camera and a personal computer-based image analysis system (Lucia-Comet version 7.0).
  • • With the help of a video camera, 50-100 images were arbitrarily selected from each sample and the percentage of DNA in the Comet tail was measured accordingly.

Lactate Dehydrogenase Activity (LDH) Assay – Protocol

  • • The basis of LDH cytotoxicity assay is the leakage of LDH, a cytoplasmic enzyme from cells when the plasma membrane is injured. This assay is useful to detect necrosis as well [13].
  • • According to the experimental protocol, 1 x 104 LMH count cells are put in a complete culture medium, in each well of a 96-well plate coated with collagen.
  • • The cells were then incubated for 24h at 37°C in 5% CO2 to set aside for attachment to the collagen-coated surface.
  • • The following day, the medium was aspirated and 200 pL of apiece concentration of the test compound in Waymouth’s medium without FBS, was added to each well in eight repeats.
  • • The control samples consist of cells without any test agent. The cells were then incubated in a CO, incubator at 37°C in 5% CO, for 24 h for DMA and 48 h for ammonia and TMA.
  • • The final test concentrations were selected based on IC50 values; 0.004-1.0% for DMA and ammonia and 0.004%-1.0% for TMA.
  • • The concentrations were investigated, and the times of incubation were chosen to detect IC50 values [10]. The viability of the cells was then measured by MTT and PrestoBlue assays.
  • • In this protocol, the cytotoxic redolent compounds can be examined, along with the check for the mechanism of cell damage (loss in cell membrane integrity and probably necrosis).
  • • The assay was then conducted with a Cytotoxicity Detection KitPLUS, as per the manufacturer’s instructions.
  • • Three control samples were included: background control (assay medium), low control (untreated cells) and high control (maximum LDH release).
  • • For determining the experimental absorbance values, the average absorbance values of the eight repeat samples and controls were calculated and then subtracted from the absorbance values of the background control.
  • • The cytotoxicity was determined by the following formula:

cytotoxicity (%) = {(exp. Value-low control)/(high control-low control)} x 100

  • • The absorbance was detected at 490 nm using a microplate reader.
  • • Results were then presented as mean ± standard deviation (SD).
  • • The mean error of the method was up to 10%.

Calculation of IC50 – Protocol

  • • IC50 is the concentration of the test compound requisite to decrease the cell survival rate to 50% of the control. These values are used as an extent of cellular compassion to a given treatment.
  • • IC50 value is determined by the following formula:

IC50=(A-B)/(A-A1)x(Ca1-Ca) + Ca,


A is a 50% decrease in viability;

A is % of viability > B;

A, is % viability < B;

CA is the concentration of the compound for A and

CA1 is the concentration of the compound for A, [10,14].

Fluorescence Microscopic Analysis –Protocol

  • • Nuclear changes were observed by employing eight-well Lab Tek™ Chamber Slides in LMH cells in the presence of test compounds.
  • • Proceeding for the culture, the slides were coated with collagen I, as per the instructions provided by the manufacturer.
  • • LMH cells were kernelled on to each well with a concentration of 2.5 x 105 cells per well. The cytotoxic redolent compounds were selected (Ammonia, DMA and TM A) to check for the mechanism of cell death, damage or apoptosis using DAPI (4',6-diamidino-2-phenylindole) staining.
  • • The final test concentrations were determined, as per the literature it is 0.03% for ammonia, DMA and TMA, which are close to the IC50 values estimated by employing MTT assay after 48 h of incubation [10].
  • • After exposure, medium with compounds was removed, cells were washed with PBS (phosphate buffer saline; pH 7.2) and fixed with 80% ethanol (for 20 min at room temperature).
  • • After air-drying, the cells were stained with 1 pg/mL DAPI in the dark. The morphology of cells was then observed at lOOOx magnification under a fluorescent microscope which is connected to a digital camera and analysed using NIS-elements BR 3.0 imaging software.
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