Research Article

 

In vivo Study of Mutagenic Activity of the Complex Antibacterial Medication of Mastitis in Mice Model

 

Sergey V. Shabunin, Galina A. Vostroilova, Liliya V. Cheskidova, Dmitriy I. Shabanov, Anastasiya A. Korchagina*

All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy, 394087, 114B, Lomonosova Str, Voronezh, Russia.

 

Received | May 06, 2021; Accepted | June 30, 2021; Published | August 15, 2021

*Correspondence | Anastasiya A. Korchagina, All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy, 394087, 114b, Lomonosova str, Voronezh, Russia; Email: [email protected]

Citation | Shabunin SV, Vostroilova GA, Cheskidova LV, Shabanov DI, Korchagina AA (2021). In vivo study of mutagenic activity of the complex antibacterial medication of mastitis in mice model. Adv. Anim. Vet. Sci. 9(10): 1601-1607.

DOI | http://dx.doi.org/10.17582/journal.aavs/2021/9.10.1601.1607

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2021 Shabunin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Mastitis of cattle remains one of the main problems of dairy farming (Pal, 2018; Ruegg and Petersson-Wolfe, 2018). The introduction of antibacterial drugs into the affected lobes of the udder, which ensures that the drug enters the area of the inflammation focus and affects the cause of the inflammatory process, is considered to be the most common way of treatment for this disease (Royster and Wagner, 2015; Hyde et al., 2019).

 

Currently, complex drugs that combine antimicrobial and anti-inflammatory effects are widely used in veterinary medicine (Reader et al., 2020). Drugs for the treatment of cow mastitis that contain β-lactam antibiotics in combination with corticosteroids, as a rule, occupy a large part of market. Medications for intramammary administration often use substances from the group of penicillins (penicillin, ampicillin, cloxacillin, amoxicillin) and prednisolone as active agent (Sipka et al., 2013; Royster and Wagner, 2015).

 

The assessment of the mutagenicity of a drug should be carried out in case of suspicion of genotoxicity of at least one of its substances, however, the complex effect of β-lactams and corticosteroids on the animal organism has not been studied enough (Turkez et al., 2017; Ahmad et al., 2018; Hartwig et al., 2020). However, it is known that the use of several substances in combination as one drug can increase the likelihood of side effects. In this regard, within the framework of this study, we set the goal of identifying the potential mutagenic effects of the medication containing semisynthetic penicillin, aminopenicillin and a glucocorticosteroid. The study of mutagenicity was carried out by accounting for chromosome aberrations in bone marrow cells and calculating the frequency of micronuclei in polychromatophilic erythrocytes in the blood of outbred mice. Since the genotoxicity of some substances can manifest itself with the accumulation of a drug in the organism after repeated admission, we evaluated the mutagenic effect of a complex drug when simulating a course treatment in mice.

 

MATERIALS AND METHODS

 

Ethical approval

The experiments on animals were approved by the Ethics Commission of All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy (Protocol No. 02-06-2020).

 

Medication

The complex medication for the treatment of mastitis in cows during lactation, containing amoxicillin trihydrate –.0%, cloxacillin sodium –5.0%, prednisolone -0.3% and excipients.

 

Animals

Detection of the potential mutagenicity of a complex antibacterial medication were conducted at the Department of Experimental Pharmacology on white outbred mice (n=42). For the experiment, we used 3 month old females with a weight of 22-24 g of vivarium breeding of “All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy”. All the works were carried out in accordance with the Guidelines for conducting preclinical studies of drugs (Durnev et al., 2012).

 

The experimental animals were kept in standard vivarium conditions (T air +18-23°C, relative humidity 45-60%). Ad libitum access to water and feeds. The manipulations with the animals in the framework of the experiment were carried out in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986).

 

Experimental design

The animals from group I (n=12), which was a negative control, were subcutaneously injected with 0.1 ml of vaseline oil for 7 days with intervals of 24 hours. The animals from group II (n=12) (positive control) were once intraperitoneally injected with cyclophosphamide (Baxter, Germany) at a dose of 20 mg/kg of body weight (Durnev et al., 2012). Mice from the experimental group III (n= 12) were injected with the complex antibacterial drug at a dose of 80 mg/kg, similarly to the animals in the negative control group. The mice of the experimental group IV were once subcutaneously injected with the drug at a 7-fold increased therapeutic dose of 560 mg/kg (n=6).

 

Frequency of chromosome aberrations

We carried out a study of the frequency of chromosome aberrations in bone marrow cells of mice in 6 animals in each group. Twenty-four h after the last injection of the drugs, the animals were euthanized by cervical dislocation; 2.5 h before this, they were injected with 0.025% colchicine solution (PanEko, Russia). Then, bone marrow cells were removed from the femur, and the resulting cell suspension was incubated for 25 min. in 0.075 M hypotonic KCl solution at 37 °C, then the cells were fixed with acetoalcohol (methanol: acetic acid - 3:1) and bone marrow cell samples were prepared with Romanowsky-Giemsa staining (Preston et al., 1987). The samples were viewed using BIOSKOP-1 microscope (× 1000; LOMO, Russia). We counted at least one hundred metaphase plates per animal. The frequency of cells with chromosome aberrations was determined, single and paired fragments, chromatid and chromosome exchanges, achromatic gaps and breaks along the centromere, as well as the cells with multiple pathologies (more than 5 per cell) were taken into account (Durnev et al., 2012).

 

Micronucleus test

The frequency of micronuclei was assessed in polychromatophilic erythrocytes (PCE) of the peripheral blood obtained from the tail vein of mice before drug injection and on days 2, 5, 7 and 9 after the start of the experiment in groups I and III, as well as on day 2 in group II (n=6). For the animals of group IV, the study of the frequency of micronuclei was not carried out (Mavournin et al., 1990; Durnev et al., 2012). The sample preparation of the peripheral blood of mice was carried out by a conventional method with the staining of blood smears according to Romanowsky-Giemsa. The samples were viewed using BIOSKOP-1 microscope (× 1000; LOMO, Russia); as micronuclei, we took into account rounded uniformly colored clearly outlined objects lying in the polychromatic erythrocytes (PCE) plane (Heddle and Salamone, 1981). The number of micronuclei was taken into account in 2000 PCE per animal, and the ratio of the number of PCE to normochromatic erythrocytes (NCE) was also taken into account (Durnev et al., 2012).

 

Statistical analysis

Statistical analysis was carried out using the statistical software package STADIA 8.0 (MSU, Russia). Values (Mean ± SE) were considered significantly different at P < 0.05.

 

RESULTS AND DISCUSSION

 

Accounting for chromosome aberrations in the bone marrow cells of mice is one of the main highly sensitive methods for studying the mutagenic effect of substances. In our study, the introduction of cyclophosphamide induced a twelvefold increase in the frequency of cells with chromosome aberrations to 12.0±1.62% (at P<0.0005 relative to the negative control), primarily due to a significant increase in the number of single fragments, which was more than 18 times higher than their number in the negative control, paired fragments were also encountered by 11 times more often and the number of exchanges was 9 times more frequent.

 

The administration of the studied medication to animals did not cause an increase in the proportion of bone marrow cells with chromosomal abnormalities (Figure 1). According to the data obtained, the proportion of the cells with chromosome aberrations in animals of the experimental group III did not differ significantly from the negative control mice (Table 1). The frequency of the cells with chromosome abnormalities in animals after a single injection of the medication at a 7-fold therapeutic dose also did not have statistically significant differences relative to the negative control group. These findings were as consistent with the studies of İstifli and Topaktaş (2010), where amoxicillin in human lymphocyte cell culture even at high doses (1 mg/ml) did not induce cytogenetic instability determined by the methods of accounting for chromosome aberrations, sister chromatid exchanges and micronuclei.

 

 

Another study demonstrated a dose-dependent increase in the frequency of chromosome aberrations in the Allium cepa test system of widely used glucocorticoids, where prednisolone was studied at doses of 5 mg/L, 20 mg/L and 60 mg/L (De Oliveira et al., 2017).

 

The micronucleus test is the most commonly used genotoxic test in in vivo studies due to its simplicity and accuracy (Hayashi, 2016). Micronuclei are formed as a result of the non-inclusion of whole chromosomes or their fragments into the main nucleus of the cell due to the pathologies of mitosis, obtaining their own membrane. These formations are able to persist for several divisions and act as a diagnostic sign of mutagenic factors of various nature (Araldi et al., 2015; Baderna et al., 2019).

 

It was found that the clastogenic effect of cyclophosphamide manifested itself in a significant increase (P<0.002) in the proportion of cells with micronuclei relative to the indicators of other groups up to 9.9±1.69 ‰. In animals from the positive control group, we found PCE containing from one to five micronuclei.

 

At the same time, the introduction of the complex drug did not induce an increase in the number of polychromatophilic cells with micronuclei in the blood of mice (Figure 2). The proportion of the cells with micronuclei in the experimental group and the negative control group did not have statistically significant differences (Figure 3; Table 2). Single micronuclei were mainly observed in the cells.

 

 

 

Table 1: Drug cytogenetic activity in the mouse bone marrow cells.

 

Groups	The number of metaphases	Percentage of pathologies per 100 cells					Total percentage of cells with pathologies
		Gaps	Single fragments	Paired fragments	Exchanges	Multiple pathologies
Group 1	600	0.7±0.23	0.7±0.23	0.2±0.18	0.2±0.18	0.0	1.2±0.69
Group 2	600	0.5±0.24	13.0±0.89***	2.2±0.66*	2.8±0.44**	0.3±0.23	12.0±1.62**
Group 3	600	0.8±0.44	0.7±0.23	0.3±0.23	0.0	0.2±0.18	1.8±1.57
Group 4	600	0.8±0.34	0.5±0.24	0.2±0.18	0.3±0.23	0.3±0.23	2.0±1.00

Data represented as Mean ±SE; changes are considered to be statistically significant at *, P<0.02; **, P<0.0005; ***, P<0.00001 relative to the parameters of the group 1.

 

Table 2: MNPCE frequency in the mouse blood 48 h after the final injection.

 

Groups	MNPCE frequency per 1000 PCE, ‰	PCE/NCE ratio, %
Negative control	1.3±1.14	2.1±0.63
Positive control	9.9±1.69*	3.1±1.10
Study drug	1.8±1.10	2.1±0.60

Data represented as Mean ±SE; changes are considered to be statistically significant at *, P<0.002 relative to the parameters of the negative control.

 

Thus, our data demonstrate no changes in the frequency of cells with chromosome aberrations in the bone marrow and PCE with micronuclei in the peripheral blood of mice who were subcutaneously injected with a combination of β-lactam antibiotics with a glucocorticosteroid for 7 days at a dose of 80 mg/kg of body weight per day and a single administration of a 7-fold daily therapeutic dose, relative to the indicators of animals in the negative control group.

 

Numerous facts indicate the safety of wide ranges of therapeutic doses of β-lactam antibiotics, which is one of the reasons for their popularity (Bozcal and Dagdeviren, 2017). A number of studies are devoted to the mutagenic potential of β-lactam antibiotics and steroidal anti-inflammatory drugs (Zavarise et al., 1984; Stemp et al., 1989; Arabski et al., 2005; İstifli and Topaktaş, 2010; De Oliveira et al., 2017). However, in publications discussing the negative and positive effects of penicillins and glucocorticoids, there is no data on their interaction (Coondoo and Chattopadhyay, 2013; Van Matre et al., 2018).

 

According to the literature data, β-lactam antibiotics can have a genotoxic effect on mammalian cells through free radical oxidation of nucleic acid molecules with reactive oxygen species (ROS) (Bozcal and Dagdeviren, 2017), as well as inhibition of the work of DNA polymerase (Metovic et al., 2013).

 

The ROS-induced DNA damage of lymphocytes and cells of the human gastric mucosa with amoxicillin at a dose of 5 mM/L after its cellular activation was shown using the DNA-comet method. At the same time, the authors believe that the used concentration of the antibiotic is achieved in the stomach only after oral administration and significantly exceeds the possible dose in the blood (Arabski et al., 2005). In addition, the consequences mediated by oxidative stress or other factors of the drug genotoxicity can, to some extent, be leveled out by the reparative systems of the organism (Christmann and Kaina, 2013). Thus, amoxicillin-induced damage to the DNA of lymphocytes was completely restored in 60 minutes (Arabski et al., 2005). It is possible that additional ROS formed during the cellular activation of penicillins (in particular, amoxicillin and cloxacillin) are successfully eliminated by the organism’s antioxidant systems (Deavall et al., 2012).

 

Zavarise et al. (1984) on the culture of human lymphocytes in vitro using methods of accounting for chromosome aberrations and sister chromatid exchanges have detected that cloxacillin exhibited clastogenic properties only in high concentrations and did not cause chromosome damage at therapeutic doses.

 

In the study by de Sousa et al. (2019), amoxicillin did not exhibit genotoxicity at doses of 5.13 × 10−3, 10.26 × 10−3, 20.52 × 10−3, 41.05 × 10−3 mM in the micronucleus test on Tradescantia pallida, which was consistent with our results.

 

We found that the PCE content in the blood did not have statistically significant differences in all the studied groups and did not go beyond the reference values (1-6%) (O’Connell, 2015). The presented data demonstrate the absence of mutagenicity, or its insignificant manifestation in the study of doses of antibiotics exceeding the daily and course doses of amoxicillin -2.4 mg/kg and 16.8 mg/kg and cloxacillin -4.0 mg/kg and 28.0 mg/kg used in the experimental groups.

 

Glucocorticosteroids can also be mutagenic due to their ability to penetrate into the cells, interact with DNA and regulate gene expression. Structural chemical similarity and mechanism of action suggest slight differences in the manifestation of genotoxicity between steroid drugs (De Oliveira et al., 2017). Thus, a number of synthetic steroids exhibited mutagenic effects in the Ames test. The authors of the study suggest that the genotoxic effect of the drugs is associated with free radical damage to DNA cells (Islam and Ahmad, 1991). At the same time, the Japanese Food Safety Commission published the results of the study in 2016 in which no mutagenic effects of prednisolone were found in vivo (Food Safety Commission of Japan, 2016). Since in some in vitro studies it showed genotoxicity, there was detected the minimum dose of 0.25 g/kg of body weight per day at which adverse effects were observed.

 

The study conducted by Hayes et al. (2013) showed that prednisolone increased micronuclei in PCE of rat bone marrow at doses of 500, 1000, 1500 mg/kg when orally administered, however, in our study, this trend was not observed, probably due to the use of smaller doses and route of inoculation.

 

The concentration of prednisolone introduced by us to the animals of groups III and IV was lower than the detected minimum toxic dose by more than 1000 times (0.24 mg/kg) and 148 times (1.68 mg/kg), respectively, which explained the absence of genotoxic action from the glucocorticoid component of the drug.

 

Thus, the cytotoxic effects observed in some cases caused by β-lactam antibiotics and glucocorticoid drugs were manifested primarily in bacterial, plant or animal models in vitro, and to a lesser extent were found in animal models in vivo, which was probably due to the differences in model objects, the effect of reparative systems and the metabolism of the investigated substances at the level of the organism (Musgrove and Camps, 2012). In addition, it should be noted that the high doses of substances used in the aforementioned sources, which significantly exceed the content of active substances in the complex drug under study. The combination of glucocorticoid and β-lactam antibiotics did not increase the mutagenicity of the composition used. Despite the increasing popularity of in vitro genotoxic tests, largely related to ethical issues (Hayashi, 2016), in our opinion, the study of mutagenicity in in vivo models is mandatory when studying the safety of new veterinary drugs due to the similarity of the organisms in laboratory and farm animals.

 

CONCLUSIONS AND RECOMMENDATIONS

 

As a result of our studies, we found that there was no mutagenic effect of the new complex medication containing prednisolone and antibiotics of the penicillin group (amoxicillin and cloxacillin) on the body of white mice when used at a 7-fold daily therapeutic dose once and for 7 days at a therapeutic dose. This is confirmed by the absence of a significant increase in the proportion of bone marrow cells with chromosome aberrations and the number of polychromatophilic blood erythrocytes with micronuclei relative to the values of the negative control group. Probably for the manifestation of the mutagenic effect of the complex medication, it is necessary to use higher doses and longer duration of administration, which requires further study.

 

ACKNOWLEDGEMENTS

 

Authors would like to thank Anna Belenko for English language consulting.

 

NOVELTY STATEMENT

 

As a result of our studies, we have found that there is no mutagenic effect of a complex antibacterial medication containing antibiotics of the penicillin group (amoxicillin and cloxacillin) and prednisolone on the organism of white mice when used subcutaneous for 7 days at a dose of 80 mg/kg of body weight and single administration at a dose of 560 mg/kg of body weight. This is confirmed by the absence of a significant increase in the proportion of bone marrow cells with chromosome aberrations and the number of blood polychromatophilic erythrocytes with micronuclei relative to the values of the negative control group.

 

AUTHOR’S CONTRIBUTION

 

All authors contributed to the study conception, design, material preparation, investigations, data collection and analysis, participated in writing the manuscript and approved the final version.

 

Conflict of interest

 

The authors have declared no conflict of interest.

 

REFERENCES

 

• Ahmad MH, Fatima M, Hossain M, Mondal AC (2018). Evaluation of naproxen-induced oxidative stress, hepatotoxicity and in-vivo genotoxicity in male Wistar rats. J. Pharm. Anal., 8(6): 400-406. https://doi.org/10.1016/j.jpha.2018.04.002

• Arabski M, Kazmierczak P, Wisniewska-Jarosinska M, Poplawski T, Klupinska G, Chojnacki, DrzewoskiJ,Blasiak J (2005). Interaction of amoxicillin with DNA in human lymphocytes and H. pylori-infected and non-infected gastric mucosa cells. Chemico-Biol. Interact., 152(1): 13-24. https://doi.org/10.1016/j.cbi.2005.01.004

• Araldi RP, de Melo TC, Mendes TB, de Sá Júnior PL, Nozima BHN, Ito ET, CarvalhoRF, de SouzaEB,de Cassia Stocco R (2015). Using the comet and micronucleus assays for genotoxicity studies: A review. Biomed. Pharm., 72: 74-82. https://doi.org/10.1016/j.biopha.2015.04.004

• Baderna D, Gadaleta D, Lostaglio E, Selvestrel G, Raitano G, Lombardo A, Benfenati E (2019). New in silico models to predict in vitro micronucleus induction as marker of genotoxicity. J. Hazard. Mater., 385: 121638. https://doi.org/10.1016/j.jhazmat.2019.121638

• Bozcal E, Dagdeviren M (2017). Toxicity of β-lactam antibiotics: pathophysiology, molecular biology and possible recovery strategies. In: Poisoning: from specific toxic agents to novel rapid and simplified techniques for analysis. Intech. Open, London, pp. 87-105. https://doi.org/10.5772/intechopen.70199

• Christmann M, Kaina B (2013). Transcriptional regulation of human DNA repair genes following genotoxic stress: Trigger mechanisms, inducible responses and genotoxic adaptation. Nucl. Acids Res., 41(18): 8403-8420. https://doi.org/10.1093/nar/gkt635

• Coondoo A, Chattopadhyay C (2013). Drug interactions in dermatology: What the dermatologist should know. Indian J. Dermatol., 58(4): 249-254. https://doi.org/10.4103/0019-5154.113928

• De Oliveira VPC, de Sousa GR, de Araujo MPT, Carneiro LC, de Andrade DML, Porto HKP, de Oliveira Santos M, de Moraes Filho AV (2017). Evaluation of the mutagenic potential of glucocorticoids by Allium cepa. Res. J. Mut., 7(1): 1-7.

• De Sousa FA, Morais CR, Vieira JS, Maranho LS, Machado FL, Pereira S, Barbosa LC, Coelho HE, Campos CF, Bonetti AM (2019). Genotoxicity and carcinogenicity of ivermectin and amoxicillin in vivo systems. Environ. Toxicol. Pharmacol., 70: 103196. https://doi.org/10.1016/j.etap.2019.103196

• Deavall DG, Martin EA, Horner JM, Roberts R (2012). Drug-induced oxidative stress and toxicity. J. Toxicol., 2012: 645460. https://doi.org/10.1155/2012/645460

• Durnev AD, Guskova TA, Yu A, Revazova VA, Merkulov OL, Verstakova VS, Zhurkov LP, Sycheva AK, Zhanataev V, Yurchenko V (2012). Guidelines for assessing the mutagenic properties of medicinal substances. In: Guidelines for conducting preclinical studies of medicaments. Grief and K, Moscow, pp. 94-114.

• Food Safety Commission of Japan (FSC) (2016). Prednisolone. Food Saf., 4(2): 52-53. https://doi.org/10.14252/foodsafetyfscj.2016010s

• Hartwig A, Arand M, Epe B, Guth S, Jahnke G, Lampen A, MartusHJ, MonienB, RietjensIMCM., Schmitz-Spanke S, Schriever-SchwemmerG, SteinbergP, EisenbrandG (2020). Mode of action-based risk assessment of genotoxic carcinogens. Arch. Toxicol., 94(6): 1787-1877. https://doi.org/10.1007/s00204-020-02733-2

• Hayashi M (2016). The micronucleus test most widely used in vivo genotoxicity test. Genes Environ., 38: 18. https://doi.org/10.1186/s41021-016-0044-x

• Hayes JE, Doherty AT, Coulson M, Foster JR, Cotton PT, O’Donovan MR (2013). Micronucleus induction in the bone marrow of rats by pharmacological mechanisms. I: glucocorticoid receptor agonism. Mutagenesis, 28(2): 227-232. https://doi.org/10.1093/mutage/ges076

• Heddle JA, Salamone MF (1981). The micronucleus assay. I. In vivo. In: Short-term tests for chemical carcinogens. Springer, New York, NY, pp. 243-249. https://doi.org/10.1007/978-1-4612-5847-6_21

• Hyde R, Tisdall D, Gordon P, Remnant J (2019). Reducing antimicrobial use on dairy farms using a herd health approach. Practice, 41(8): 368-382. https://doi.org/10.1136/inp.l5518

• Islam S, Ahmad M (1991). Mutagenic activity of certain synthetic steroids: Structural requirement for the mutagenic activity in Salmonella and E. coli. Mutat. Res. Genet. Toxicol., 259(2): 177-187. https://doi.org/10.1016/0165-1218(91)90051-M

• İstifli ES, Topaktaş M (2010). Cytogenetic genotoxicity of amoxicillin. Environ. Mol. Mutag., 51(3): 222-228.

• Mavournin KH, Blakey DH, Cimino MC, Salamone, MF, Heddle JA (1990). The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US environmental protection agency gene-tox program. Mutat. Res. Rev. Genet. Toxicol., 239(1): 29-80. https://doi.org/10.1016/0165-1110(90)90030-F

• Metovic A, Mackic-Djurovic M, Ibrulj S (2013). Analysis of chromosome aberrations contained in vitro human peripheral blood lymphocytes after treatment with ceftriaxone. Med. Arch., 67(4): 228-232. https://doi.org/10.5455/medarh.2013.67.228-232

• Musgrove C, Camps M (2012). Models for detection of genotoxicity in vivo: Present and future. In: Mutagenesis. Intech Open, London, pp. 31-50. https://doi.org/10.5772/50554

• O’Connell KE, Mikkola AM, Stepanek AM, Vernet A, Hall CD, Sun CC, Yildirim E, Staropoli J, Lee J, Brown DE (2015). Practical murine hematopathology: A comparative review and implications for research. Comp. Med., 65(2): 96-113.

• Pal M (2018). Mastitis: A major production disease of dairy animals. Agric. World, 4: 46-51.

• Preston RJ, Dean BJ, Galloway S, Holden H, McFee AF, Shelby M (1987). Mammalian in vivo cytogenetic assays analysis of chromosome aberrations in bone marrow cells. Mutat. Res. Genet. Toxicol., 189(2): 157-165. https://doi.org/10.1016/0165-1218(87)90021-8

• Reader J, Angel T, Rogers I (2020). Practical use of nonsteroidal anti-inflammatory drugs in farm practice. Livestock, 25(5): 202-209. https://doi.org/10.12968/live.2020.25.5.202

• Royster E, Wagner S (2015). Treatment of mastitis in cattle. Vet. Clin. North Am. Food Anim. Pract., 31(1): 17-46. https://doi.org/10.1016/j.cvfa.2014.11.010

• Ruegg PL, Petersson-Wolfe CS (2018). Mastitis in dairy cows. Vet. Clin. North Am. Food Anim. Pract., 34(3): 9-10. https://doi.org/10.1016/j.cvfa.2018.08.001

• Sipka, A, Gurjar A, Klaessig S, Duhamel GE, Skidmore A, Swinkels J, Cox P, Schukken Y (2013). Prednisolone and cefapirin act synergistically in resolving experimental Escherichia coli mastitis. J. Dairy Sci., 96(7): 4406-4418. https://doi.org/10.3168/jds.2012-6455

• Stemp G, Pascoe S, Gatehouse D (1989). In vitro and in vivo cytogenetic studies of three β-lactam antibiotics (penicillin VK, ampicillin and carbenicillin). Mutagenesis, 4(6): 439-445. https://doi.org/10.1093/mutage/4.6.439

• Turkez H, Arslan ME, Ozdemir O (2017). Genotoxicity testing: progress and prospects for the next decade. Exp. Opin. Drug Metab. Toxicol., 13(10): 1089-1098. https://doi.org/10.1080/17425255.2017.1375097

• Van Matre ET, Satyanarayana G, Page RL, Levi ME, Lindenfeld J, Mueller SW (2018). Pharmacokinetic drug-drug interactions between immunosuppressant and anti-infective agents: Antimetabolites and corticosteroids. Ann. Transplant., 23: 66-74. https://doi.org/10.12659/AOT.906164

• Zavarise G, Ghiazza G, Grillo G, Morino P, Mondavio M (1984). Evaluation of the effect of cloxacillin on human lymphocyte chromosomes through the study of karyotypic changes and sister chromatid exchanges. Bollettino Della Societa Italiana di Biologia Sperimentale, 60(11): 21-43.