1 Introduction

Fourier-Transform Infrared (FTIR) Spectroscopy is an absorption spectroscopic technique that records the interferometry of an IR light after being absorbed by the sample. The application of the FTIR technique in biological [1] sciences has flourished recently due to its rapidity, accurate optical calibration, and enhanced sensitivity [2, 3]. The molecular-level information provided by FTIR spectroscopic technique makes it one of the accurate tools that investigate functional groups, chemical bonding, and molecular conformations [4].

This technique has been used widely in biophysical and biochemical research for both qualitative and quantitative analysis of biomolecules [5]. The spectra obtained from FTIR analyzes can give distinct unique patterns for different cells that give an accurate performance for taxonomic differentiation [6]. FTIR spectroscopy could be considered the most suitable approach for diagnosing cytotoxicity and monitoring the damage induced by toxic materials in biological tissues, cells and body fluids [7].

Recent studies employed the FTIR technique as a biophysical indicator in assessing the toxic effects of various poisonous substances on different parts of the body, such as the Liver [7], Brain [8], kidney [9], heart [1], spleen [10] and blood serum [11]. Comparing biochemical changes in the IR spectra of healthy and poisoned samples is a convenient, non-destructive tool to distinguish them apart [12].

2, 4-Dichlorophenoxyacetic acid (2, 4-D) is one of the most used herbicides of the chlorophenoxy chemical family worldwide. Even though the toxicity of such herbicide is considered moderate to low, it is correlated with the time of exposure and concentration. It has been proven that 2, 4-D may cause an acute poisoning effect in humans at doses above 300 \(\mathrm{mg}{\mathrm{ kg}}^{-1}\) [13, 14]. High doses may result in developing motor incoordination, weakening of reflexes, tenseness, and coma in humans and rats [15].

Bhat et al. 2023 Studied the germination of fenugreek seeds under exposure to sodium halide salts. The germination process was conducted under Nacl, NaI and NaF aqueous solutions in 50 and 100 ppm exposure for two weeks. They observed differences in root and shoot lengths compared to the control. After separating and loading leaves, roots and shoots on KBr disks, FTIR spectroscopic technique was applied for analysis. Some conformational changes in macromolecules treated with sodium halide salts were indicated as unique FTIR spectral patterns were observed. They concluded that FTIR spectroscopy is a suitable technique for detecting conformational changes in molecular components in young seedlings as it takes minimal sample amounts [16, 17].

Blood serum of rats was the primary focus of the current study as the measurement of the serum's different components is the most used method for toxicity investigations as well as the diagnosis of many diseases. Furthermore, measuring serum biomarkers such as liver or heart enzymes is considered a very useful tool in research focusing on toxicity studies [18]. Also, measuring safety biomarkers in serum, for example, enables serial monitoring and could reduce the number of animals used compared to other methods, such as microscopic examination [19]. Our aim in this study is to investigate and assess the 2, 4-D-induced blood serum toxicity utilizing the FTIR technique.

2 Materials and methods

2.1 Experimental animal

In this study, a count of 15 male, albino Wister strain rats (270 ± 30 g) were obtained from King Fahd Medical Research Center. Rats were accommodated in separate cages, and each house was maintained in relative humidity of 70%, an average temperature of 25° ± 1 C and lighting for 12 h daily. The rats received the same basic diet in pellet form the Grain Silos and Flour. The standard diet comprised crude protein (20%), crude fat (4%), crude fiber (3.5%), ash (6%), salt (0.5%), calcium (1%), phosphorus (0.6%), vitamin A (20 IU/g), vitamin D (2.2 IU/g) and vitamin E (70 IU/kg). After acclimatization, the rats were divided randomly into group 1, which served as a control group (N = 5) and group two, which was treated with 2, 4-D (N = 10). Group two was treated with a single dose of 639 mg/kg body weight, which is the LD50 dose according to (EPA) (2005) [18]. Rats were sacrificed by decapitation 24 h post 2, 4-D administration. Afterwards, serum was obtained by centrifugation of the blood samples taken from each rat.

2.2 FTIR sample preparation

For the purpose of FTIR results analyzation, all serum samples were freeze-dried by Christ freeze dryer under the pressure of 0.02 mbar and -60°c until serum became fine powder and prepared on the KBr/sample disks. Samples were measured IR spectrophotometrically in triplicate using a Shimadzu FTIR-8400s spectrophotometer with a continuous nitrogen purge. For each rat, three IR spectra were obtained from different KBr disks and then coadded to produce one spectrum. Pellets were scanned at room temperature in the 3600–445 cm-1 spectral range. Background spectra, collected under identical conditions, were automatically subtracted from the sample spectra.

2.3 Statistical analysis

SPSS software was used in the current study to convert the resulting data statistically to mean ± SD. The experimental groups were tested using the Mann–Whitney test to measure the differences between normal and treated groups. Significance was based on the value p < 0.05. Two parameters were calculated and expressed as intensity (I) and area (A) ratio for the lipid region [I (2960/2929), and A (1392/2924)].

3 Results

In this study, FTIR spectroscopy was used to investigate the structural molecular changes due to the toxic effect of the pesticide 2, 4-D on Wister rats. Figure 1: Comparison of the FTIR spectra of both control and 2, 4-D groups.

Fig. 1
figure 1

Comparison of the FTIR spectra of both control and 2, 4-D groups

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Demonstrates serum samples' average normalized mid-IR spectra after spectral pretreatment for 2, 4-D, and the control group. To increase spectral overlapping bands' resolution, the Gaussian function was used for the best fit of these bands. Omnic software was utilized to apply Gaussian components. Moreover, to eliminate any artefacts which may be caused by variations in the experimental conditions, e.g., sample concentration or distribution in KBr when measured by FTIR spectrometer, the band intensity and area ratios of some specific infrared bands have been evaluated for quantitative comparison between control and treated groups [7]. Gaussian fitting, peak height ratios and spectral analysis were investigated using row FTIR data and are illustrated in Fig. 2: (a) Curve fitting of rat blood serum in the IR spectral range 3700–2700 of the control group. (b) Curve fitting of rat blood serum in the IR spectral range 3700–2700 of 2, 4-D group., Fig. 3: (a) Curve fitting of rat blood serum in the IR spectral range 1800–1500 of control group. (b) Curve fitting of rat blood serum in the IR spectral range 3700–2700 of 2, 4-D group, and Fig. 4. Both IR spectra were precisely analyzed to identify serum from 2, 4-D treated rats and healthy ones. Spectra of control and poisoned rats were compared; the results are illustrated in Tables 1 and 2.

Fig. 2
figure 2

a Curve fitting of rat blood serum in the IR spectral range 3700–2700 of the control group. b Curve fitting of rat blood serum in the IR spectral range 3700–2700 of 2, 4-D group

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Fig. 3
figure 3

a Curve fitting of rat blood serum in the IR spectral range 1800–1500 of control group. b Curve fitting of rat blood serum in the IR spectral range 3700–2700 of 2, 4-D group

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Fig. 4
figure 4

a Curve fitting of rat blood serum in the IR spectral range 900–1200 of control group. b Curve fitting of rat blood serum in the IR spectral range 900–1200 of 2, 4-D group

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Table 1 The effect of 2,4-Dichlorophenoxyacetic acid herbicide on intensities for rats' blood serum
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Table 2 The effect of 2,4-Dichlorophenoxyacetic acid herbicide on areas for rats' blood serum
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3.1 Lipids

3.1.1 Peaks 2960 cm−1, 2875 cm−1, 2929 cm−1, 1454 cm−1 and cholesterol (at 1116 cm−1, 1743 cm−1)

Intensities and areas were decreased due to the 2, 4-D stress at 2960 cm−1, 2875 cm−1, and 2929 cm−1 (Tables 1 and 2) perceptive to asymmetric vibration of C–H in CH3 (mainly lipids), symmetric stretching vibrations of C–H of protein and lipid, and asymmetric vibration of C–H in CH2 (long-chain fatty acids), respectively [11, 19]. There was also a reduction in the intensity and area at 1454 cm−1 (Tables 1 and 2) associated with C-H scissoring bending vibration, mainly lipid [20]. Furthermore, there was a reduction in the intensity and the area of 1743 cm−1 (Tables 1 and 2) attributed to the C = O group of cholesterol ester (HDL) for the 2, 4-D treated rats. This is consistent with the reduction in area and intensity for the intoxicated group at 1116 cm−1 (Tables 1 and 2) attributed to cholesterol [21]. The changes in the absorption peak ratio were calculated for the serum samples of the control and the intoxicated rats at 1392/2924 for the relative content of cholesterol esters from [22]. The result shows a reduction caused by toxicity from 0.9187 ± 0.07221 in control to 0.8895 ± 0.03647 in 2, 4-D, consistent with the decrease observed in cholesterol intensity and area at 1743 and 1116 cm−1. Our data also revealed a reduction in the intensity peak ratio for the lipid \(2961/2846\) in the 2, 4-D treated group from 1.55 ± 0.005 to 1.49 ± 0.01 in control group.

3.2 Asymmetric PO2 stretching vibration mode of nucleic acid

3.2.1 Peak at 1240 cm−1

Our results indicate an increase in the area for the 2, 4-D compared to the control group at 1240 cm−1 attributed to the Asymmetric PO2 stretching vibration mode of nucleic acid. The results are stated in (Table 2).

3.3 Glucose

3.3.1 Peak at 1078 \({cm}^{-1}\)

For the region 1000–1200 \({\mathrm{cm}}^{-1}\), the peak intensity and area at 1078 \({\mathrm{cm}}^{-1}\) wavenumber due to vibrations of C-O characterization stretching of glucose [21] decreased as a result of the toxicity induced by 2, 4-D (Tables 1 and 2).

3.4 Protein and secondary structure

3.4.1 Peaks at 2875 \({cm}^{-1}\) and 2925 \({cm}^{-1}\)

Our results indicate that the intensities of both protein peaks (2875 and 2925 \({\mathrm{cm}}^{-1}\)) were decreased by toxicity for the poisoned group (Table 1).

3.4.2 Peaks at 1646 \({cm}^{-1}\) and 1691 \({cm}^{-1}\)

The secondary structure of protein was altered by toxicity. Alpha helix secondary structure area at 1646 \({\mathrm{cm}}^{-1}\) was decreased by toxicity from 116.1423% to 114.9327%, while beta-sheets area at 1691 \({\mathrm{cm}}^{-1}\) was increased by toxicity from 77.5693 to 79.7561%.

4 Discussion

4.1 Lipids

Our data indicated a reduction in the peaks 2960, 2875, 2929, 1454 cm−1 corresponding to lipids and cholesterol at 1116, 1743 cm−1. This decrease in lipids and cholesterol could be indicative for liver damage as stated in [23]. The study showed that during liver damage, there was a reduction by 50 % in cholesterol and other lipids in 2 and 4-days post Praseodymium administration [23]. Another reason for this decrease could be attributed to acetic acid content in the 2, 4-D herbicide that reduced serum total cholesterol as described by [24]. Liver damage might also cause inhibition of lipogenesis in the liver, which can be responsible for such reduction in lipid and cholesterol as was proved by Dakhakhni et al. [7].

Oxidative stress was proven to result from the accumulation of reactive oxygen species (ROS). ROS is produced by environmental factors, such as pollutants like pesticides. Oxidative stress leads to the degradation of serum carbohydrates, nucleic acids, lipids and protein [25], and alters their function. ROS results in lipid peroxidation by pulling out an electron from polyunsaturated fatty acids because of the presence of unpaired electrons in ROS structure. Also, disruption of the membrane lipid bilayer arrangement may occur, which could alter the permeability of the cell membrane [26].

4.2 Asymmetric PO2 stretching vibration mode of nucleic acid

This increase observed could be a result of structural chromosomal damage due to a rise in molecule freedom degrees caused by chromosome fragmentation. A study in 2012 [27] has proved associated damage in DNA single and double strands as well as an alternation in DNA backbone in the range (950–1240 cm−1) after exposure to Proton Microbeam [27].

ROS has been proven to lead to DNA modifications. These modifications may involve single- or double-stranded DNA breaks, degradation of nucleic bases, mutations, purine, pyrimidine or sugar-bound modifications and deletions or translocations [26]. When the damage of DNA is not repaired, programmed cell death or accumulated mutations may occur, resulting in genomic instability, which can trigger tumorigenesis and many other genomic disorders [25, 28].

4.3 Glucose

The liver plays an important part in maintaining glucose homeostasis in blood, however sustaining normal glucose homeostasis ability by the liver, could be defeated by any disturbance in the liver’s intracellular functioning, metabolism, or structure. Sequentially the glucose secretion will be altered causing hypoglycemia [29]. The reduction at 1078 \({\mathrm{cm}}^{-1}\) that is associated with glucose could be due to several glycogen storage diseases (GSD) and/or severe liver disease [30], which is consistent to our finding in a previous study [7].

The findings of [31] revealed that there was a decrease in serum glucose levels after glyphosate oral administration. They ascribed the cause to the pesticide action as a stress factor which causes hypoglycemia. Also, [32] stated that the Hypoglycemic effect induced by 2, 4-D oral application could be due to herbicide's direct hypoglycemic effect or by affecting β-pancreatic cells. Thus, the failure of β-cell leads to no decrease in the secretion of insulin by β-cell and, in turn no α-cell glucagon secretion increases during hypoglycemia.

4.4 Protein and secondary structure

Decreases at Peaks 2875 and 2925 \({\mathrm{cm}}^{-1}\) for the poisoned groups could be attributed to protein expression depression in the liver. Jiménez-torres et al. claimed that protein expression depression in the liver was noticed post a single dose of CCl4 [33]. It is well known that ROS affects proteins in various ways such as alteration of the electrical charge of proteins, fragmentation of the peptide chain, oxidation of specific amino acids and cross-linking of proteins. Therefore, this could increase the susceptibility to proteolysis by specific protease degrading action. Moreover, lipid peroxidation products such as unsaturated aldehydes and MDA, can inactivate many cellular proteins by protein cross-linkage formation and thus inhibit their function. Also, disruption of the membrane lipid bilayer arrangement may occur, which may inactivate membrane-bound receptors and enzymes, causing an increase in tissue permeability with which cytotoxicity is mediated [25].

Furthermore, the reductions occur at 1646 and 1691 \({\mathrm{cm}}^{-1}\) peaks are consistent with [34], who stated that 2, 4-D and other pesticides can cause an increase in β-sheets and a reduction in α-helix [34]. This could be explained as follows: the structure of β-sheet content in proteins is usually formed by salt-, solvent- or thermal-induced aggregation due to protein denaturation. Findings from the treated group suggest that the increase in the interactions of intermolecular hydrogen-bond results in a higher molecular weight aggregation formation، which are β-sheet structures, so the the secondary structure of proteins is transformed from alpha-helix to β-sheets [8]. This alteration in turn could impair the function of the affected protein. Another study [35] stated that the FTIR spectroscopy results proved α-helix transformation into beta-sheet. This was shown by the α-helix percentage reduction and the β-sheet percentage rise due to hydroxyapatite crystal formation [35, 36]

5 Conclusion

In the current study, FTIR spectroscopic technique was applied to investigate the effect of 2, 4-D on dried blood serum samples. Spectra comparisons have shown changes in lipid, DNA, and glucose content in 2, 4-D treated samples compared to healthy ones. Furthermore, this technique exhibited a rapid and precise performance in identifying alternations in protein content and conformational structure in response to herbicide intoxication. Based on FTIR spectroscopic technique's sensitive, reliable, and accurate findings, it is recommended that more environmental pollutants' effects on different organs be evaluated.