What is considered cardiotoxicity of anthracyclines in animal studies.

Anthracyclines are commonly used anticancer drugs with well‑known and extensively studied cardiotoxic effects in humans. In the clinical setting guidelines for assessing cardiotoxicity are well‑established with important therapeutic implications. Cardiotoxicity in terms of impairment of cardiac function is largely diagnosed by echocardiography and based on objective metrics of cardiac function. Until this day, cardiotoxicity is not an endpoint in the current general toxicology and safety pharmacology preclinical studies, although other classes of drugs apart from anthracyclines, along with everyday chemicals have been shown to manifest cardiotoxic properties. Also, in the relevant literature there are not well‑established objective criteria or reference values in order to uniformly characterize cardiotoxic adverse effects in animal models. This in depth review focuses on the evaluation of two important echocardiographic indices, namely ejection fraction and fractional shortening, in the literature concerning anthracycline administration to rats as the reference laboratory animal model. The analysis of the gathered data gives promising results and solid prospects for both, defining anthracycline cardiotoxicity objective values and delineating the guidelines for assessing cardiotoxicity as a separate hazard class in animal preclinical studies for regulatory purposes.

Introduction

Chemotherapeutics cardiotoxicity is a major concern for clinicians treating different kinds of cancer, as it seriously affects their treatment options and the survival of the patient. The cut-off values for the identification of cardiotoxicity caused by chemotherapeutics in humans differ between the American and European guidelines: the definition considers a lower cut-off value of normality for the left ventricular ejection fraction (LVEF) of 50% in Europe (1) and 53% in the USA (2). Both Guidelines emphasize that a drop of LVEF compared to the patient's previous values is also required. This definition is crucial for patients and clinicians, as patients presenting this decline in cardio-imaging indices of cardiac function should be treated with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) in combination with β-blockers (3); nevertheless, modifications of anticancer treatment in such patients remain a matter of discussion among different specialists.

In animal studies, where new anticancer substances are evaluated and different agents are tested to overcome anticancer drugs cardiotoxicity, identification of the extent of cardiotoxicity is crucial and necessary for the evaluation of any favourable effects of the counteracting agent (4). In this regard, cardiac imaging is more often used at analogy to the clinical setting. Biomarkers and clinical signs of heart failure are also taken into consideration, but cardiac imaging in animal studies has gained momentum.

Anthracyclines are a class of drugs used in cancer chemotherapy isolated from Streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, as well as breast, stomach, uterine, ovarian, bladder cancer, and lung cancers (5–7). The first anthracycline discovered was daunorubicin (trade name Daunomycin), which is produced naturally by Streptomyces peucetius, a species of actinobacteria. Clinically, the most important anthracyclines are doxorubicin, daunorubicin, epirubicin and idarubicin. Anthracyclines, which are considered as well-established cardiotoxic compounds causing myocardial suppression in a considerable number of patients, are also used in animal studies as an easy and low-cost method to introduce a model of dilated cardiomyopathy (8), as opposed to interventional research animal models of infarction and myocardial ischaemia [e.g., permanent ligation of the left anterior descending artery (LAD) or cryo-pen application on the surface of the heart leading to cryo-scar ischemia]. Different animal species and various anthracyclines dosing and administration schemes have been applied in the literature for the development of anthracyclines cardiotoxicity (9) and monitoring of the progress thereof, as well as testing different compounds/schemes for ameliorating myocardial damage. To monitor cardiotoxicity caused by anthracyclines, cardiac imaging is primarily used and secondarily, biochemical markers.

At the same time, other pharmaceutical compounds, such as anabolic steroids, along with everyday chemicals, such as metals and pesticides, have been implicated to adversely affect cardiac pathology causing function impairment (10). Toxicity and risk for human health posed by chemicals are well controlled at a European level through a thoroughly developed regulatory network. Nevertheless, cardiotoxicity is not described as a separate hazard class and no specific classification criteria are available in order to legally classify chemicals well in advance as cardiotoxic and avoid potential long-term cardiovascular complications, which could significantly burden any national health system.

But, what is considered cardiotoxicity of anticancer agents and specifically anthracyclines when parameters of cardiac imaging are monitored in animal studies? Is there a uniformity in animal models of anthracyclines cardiotoxicity induction and most importantly, do all studies describe the same decline of myocardial function? Addressing these issues could be of wider use both in clinical medicine and practice, when assessing agents employed for salvation to cardiotoxic complications during oncology treatment, for example, as well as to regulators, when trying to establish reference values in echocardiographic function representing cardiotoxicity induced in animals by chemicals.

In the current in depth review, the identification of most commonly used metrics of myocardial function in animal studies of anthracycline induced cardiotoxicity are presented, along with the range of these values differentiating normal cardiac function from animals with pathological echocardiographic findings indicative of anthracycline cardiotoxicity as per author presentation.

Materials and methods

PubMed electronic database was systematically searched to detect all original research studies published until March 1, 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement (11). The specific literature search strategy used was: [AND (“*rats*” OR “*doxorubicin* OR “*echocardiography*” OR “anthracycline” OR “*ejection fraction*”)] either in the Title, or the Abstracts. The reference list of the retrieved studies was further evaluated for the relevance of the subject and the eligibility by screening the titles/abstracts of full papers. The non-English citations (<5) were reviewed separately. Animal data only from rat species were assessed, as it is evident from the search string. All types of citations other than original research studies (e.g., review articles) were excluded. Two authors (NG and CT) independently assessed the title and the abstract content (or both) of each record retrieved to decide which studies should be further evaluated and extracted all data. Disagreements were resolved through consensus or by consultation with a third author (KT). A final draft of the manuscript was prepared after several revisions and approved by all authors. In total, 86 published manuscripts on animal studies were considered for the systematic review (Fig. 1).

Figure 1.

Prisma flow chart (literature search) for the present study design.

Despite the small size of the rat heart and the fast heart rate, echocardiography is systematically used in the evaluation of rat heart function (12). Data for 2 main indices of LV contractility were extracted from the list of studies.

The first index is LV fractional shortening (FS) and is calculated by the formula: FS (%) = [LV end-diastolic diameter (LVDd) minus LV end-systolic diameter (LVDs)]/LVDd × 100.

LVEF is the second and more common, index of LV contractility. EF can be calculated from the equation: EF (%) = [(LVDd3 - LVDs3) / LVDd3] × 100 (13) or from the equation: EF (%) = (LVEDV-LVESV)/LVEDV × 100, where LVEDV is the LV end-diastolic volume and LVESV is LV end-systolic volume (12).

Results

A summary of the studies reviewed in the present report is presented in Table I.

Table I.

Treatment protocol and main findings of the studies that examined anthracyclines cardiotoxicity in rats reviewed in the present report.

Publication	No. of animals/rat strain/sex	Anthracycline administered	Anthracycline total dose	Duration	Summary of findings	Calculations
Zhang et al (14)	30/Sprague Dawley	Doxorubicin	1 mg/kg	Daily doses	Cardiac dysfunction	Values calculated
	rats/male	(brand name Adriamycin)		for 2 weeks	(parameters monitored:	manually by the
					diastolic left ventricular	authors of this
					internal dimension, systolic	review
					left ventricular internal
					dimension, LVEF and LVFS)
Tian et al (15)	70/Sprague Dawley	Doxorubicin	3.0 mg/kg	Once a week	Cardiomyopathy	Values provided
	rats/Male			for 6 weeks		in the manuscript
Andreadou et al (16)	90/Wistar rats/male	Doxorubicin	18 mg/kg, ip	6 equal doses	Cardiomyopathy	Values provided
				for 2 weeks	(parameters monitored:	in the manuscript
					cardiac geometry, function
					and histopathology)
Oliveira et al (17)	20/Wistar rats/male	Doxorubicin	5 mg/kg, ip	Once a week	Ventricular dysfunction	Values provided
				for 4 weeks		in the manuscript
Hydock et al (18)	46/Sprague-Dawley	Doxorubicin	10 mg/kg ip	Acute	Parameters altered:	Values provided
	rats/Male			administration	LVFS and LVPWT	in the manuscript
				(bolus)
Fernandez-Fernandez	36/Sprague-Dawley rats	Doxorubicin	18 mg/kg	Over 12 days	Cardiac function altered	Values provided
et al (19)	Wistar rats Fischer-344				(LVFS, left ventricular	in the manuscript
	rats/NM				developed pressure,
					contractility and relaxation,
					cardiac capillary permeability)
Todorova et al (20)	27/Fisher 344 rats/female	Doxorubicin	12 mg/kg	Twice per week	Parameters monitored:	Values provided
			(1.5 mg/kg each)	for 4 weeks	Plasma levels of troponin I	in the manuscript
					Left ventricle (LV) function,
					LV PWT, LV volume,
					LVEF, LVFS
Vasić et al (21)	68/Wistar rats/male	Doxorubicin	15 mg/kg ip	Every other day	Parameters monitored:	Values provided
				for 2 weeks	Echocardiography,	in the manuscript
					serum cardiac troponins,
					heart rate variability and
					blood pressure variability
Mathias et al 22)	64/Wistar rats/male	Doxorubicin	20 mg/kg ip	Acute administration	Altered LVFS	Values provided
				(a single injection)		in the manuscript
Wang et al (23)	40/Sprague-Dawley	Doxorubicin	15 mg/kg ip	Acute administration	Altered LVEF, LVFS	Values calculated
	rats/male	(brand name Adriamycin)		(a single injection)	and LV outflow	manually by the
						authors of this
						review
Arozal et al (24)	25/Sprague-Dawley	Daunorubicin	3 mg/kg/day	Every other day	Altered cardiac function	Values provided
	rats/male		(18 mg/kg total dose)	for 12 days	(haemodynamic status	in the manuscript
					and echocardiography)
Argun et al (25)	40/10-week-old	Doxorubicin	4 mg/kg/dose to	Twice a week	Parameters monitored:	Values provided
	Wistar albino rats/male		a cumulative dose	for 2 weeks	Serum BNP and C-type	in the manuscript
			of 16 mg/kg, ip		natriuretic peptide
					LV functions by
					echocardiography
					and histological
					assessment
Tatlidede et al (26)	32/Wistar albino rats of	Doxorubicin	20 mg/kg, ip	Every other day	Parameters monitored:	Values provided
	both sexes			for 2 weeks	BP and HR,	in the manuscript
					echocardiography
					Lactate dehydrogenase
Razmaraii et al (27)	24/adult Wistar rats/male	Doxorubicin	2 mg/kg/48 h	Over a 12-day period	Parameters monitored:	Values provided
					LVSP, LVDP, rate of	in the manuscript
					rise/drop of LV pressure,
					LVEF, LVFS, contractility
Gziri et al (28)	43/ pregnant Wistar	Doxorubicin	10 or 20 mg/kg i.v.	On 18th day of	Altered left ventricular	Values provided
	rats/female			pregnancy	function	in the manuscript
Oliveira et al (29)	29/adult Wistar rats/male	Doxorubicin	Accumulated doses of	Four weekly	Myocardial fibrosis	Values provided
			8 (n=8), 12 (n=7), and	injections	Altered left ventricular	in the manuscript
			16 (n=7) mg/kg, ip	over 8 weeks	systolic function
Carvalho et al (30)	64/Wistar rats/male	Doxorubicin	20 mg/kg, ip	Acute	LVEF monitored	Values provided
				administration		in the manuscript
				(a single injection)
Stewart et al (31)	72/Sprague Dawley	Doxorubicin	15 mg/kg, ip	Acute administration	Parameters monitored:	Values provided
	rats/male			(a bolus injection)	LV septal and PWT,	in the manuscript
					LVESd, LVEDd, mitral
					and aortic valve blood
					flow profiles, heart dimensions
Polegato et al (32)	35/Wistar rats/male	Doxorubicin	20 mg/kg, ip	Acute administration	Parameters monitored:	Values provided
				(a single dose)	LVFS, isovolumetric	in the manuscript
					relaxation time and
					myocardial passive stiffness
Lee et al (33)	20/Sprague Dawley rats/male	Doxorubicin	Cumulative dose:	Once every two days	Impaired LV function	Values calculated
			20 mg/kg, ip	for 6 times	and performance	manually by the
						authors of this
						review
Cheah et al (34)	29/Wistar rats/male	Doxorubicin	5 mg/kg, iv	Acute administration	Parameters monitored:	Values provided
				(a single dose)	BP, HR, LVED volume,	in the manuscript
					other echocardiographic
					parameteres
Li et al (35)	48/adult Sprague-Dawley	Doxorubicin	Cumulative dose:	Over a 4-week period	Parameters monitored:	Values provided
	rats/male		16 mg/kg, ip		serum BNP level	in the manuscript
					LVEDd, LVESd, LVEF, LVFS
Dundar et al (36)	28/adult Wistar albino	Doxorubicin	15 mg/kg, ip	Acute administration	Parameters monitored:	Values provided
	rats/female			(a single dose)	LVIDd and LVISd	in the manuscript
					via the parasternal long
					axis two-dimensional images.
					LVFS and LVEF
Barış et al (37)	31/Sprague-Dawley	Doxorubicin	25 mg/kg, ip	For 12–14 days	Parameters monitored:	Values provided
	rats/male				left ventricular ejection f	in the manuscript
					raction (LVEF), LVFS and
					mitral lateral annulus (s’)
					velocity + left ventricular
					end-diastolic and end-systolic
					diameters
Lu et al (38)	60/Sprague-Dawley	Doxorubicin	2.5mg/kg/week, ip	For 6 weeks	Parameters monitored:	Values provided
	rats/male				LVFS and LVEF	in the manuscript
O'Connell et al (39)	115/adult Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	6 doses over a period	Parameters monitored:	Values provided
			(cumulative dose	of 2 weeks	left ventricular systolic	in the manuscript
			15 mg/kg)		and diastolic dimensions and
			2 mg/kg, ip (cumulative	Once a week	EF
			dose 18 mg/kg)	for 9 weeks
Chang et al (40)	71/Sprague-Dawley rats/nm	Doxorubicin	3 mg/kg/day, iv	Once a week	Parameters monitored:	Values provided
				for 6 weeks	SWT and PWT, LVED	in the manuscript
					dimensions, LVES dimensions,
					LVEF
Teng et al (41)	46/Sprague-Dawley	Doxorubicin	2 mg/kg, ip	Once a week for 8	Parameters monitored:	Values provided
	rats/male			weeks	LVED dimensions, LVES	in the manuscript
					dimensions, FS
Kim et al (42)	61/Sprague-Dawley	Doxorubicin	1.25 mg/kg, ip	Every other day for	LV systolic/diastolic	Values provided
	rats/male			1 month (16 times)	dysfunction	in the manuscript
Kondru et al (43)	24/Wistar rats/male	Doxorubicin	2 mg/kg, ip	Once in a week	Myocardial dysfunction	Values calculated
				for 5 weeks		manually by the
						authors of this
						review
Moriyama et al (44)	66/Crl:CD(SD) rats/male	Doxorubicin	2 mg/kg, iv	Once weekly,	Parameters monitored:	Values provided
				for 6 weeks	LVEDd, LVESd, LVFS	in the manuscript
Burdick et al (45)	20/Crl:CD(SD) rats/male	Doxorubicin	2 mg/kg, ip	Once a week	Parameters monitored:	Values calculated
				for 6 weeks	LVFS	manually by the
						authors of this
						review
Ammar et al (46)	50/Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	3 times a week	Parameters monitored:	Values calculated
				for 2 weeks	LVED dimensions	manually by the
					and LVSD dimensions, FS	authors of this
						review
Calvé et al (47)	21/Sprague-Dawley	Doxorubicin	3 mg/kg	Acute administration	Parameters monitored:	Values provided
	rats/female			(on postnatal	IVSd, LVPWd, LVIDd,	in the manuscript
				day 26th)	LVISd
Shen et al (48)	150/Sprague-Dawley	Doxorubicin	1 mg/kg, ip	Twice a week	Parameters monitored:	Values provided
	rat/male		2 mg/kg, ip (cumulative	Once a week	LVESd, LVEDd, LVEF	in the manuscript
			dose 12 mg/kg)	for 6 weeks
Wu et al (49)	32/Sprague-Dawley rat/male	Doxorubicin	2.5 mg/kg, ip	Every second day	Parameters monitored:	Values calculated
			(cumulative dose	for 6 times	LVEDP, LVESP and left	manually by the
			15 mg/kg)		ventricular pressure	authors of this
					(±dP/dtmax), LVEF and LVFS	review
Shoukry et al (50)	32/Wister rats/male	Doxorubicin	2.5 mg/kg, ip	2 weeks	Parameters monitored:	Values calculated
					LVIDd, LVIDs, LVFS and	manually by the
					LVEF	authors of this
						review
Niu et al (51)	26/Sprague Dawley	Doxorubicin	Each dose consisted of	For 2 weeks on days	Parameters monitored:	Values provided
	rats/male		1, 1, 2, 2, 3, 3, 4	1st, 3rd, 5th, 7th,	IVSd, IVSs, LVPWd and	in the manuscript
			and 4 mg/kg, ip	9th, 11th, 13th and	LVPWs, LVIDd, LVIDs
			(cumulative dose	15th, respectively	were measured on left
			20 mg/kg)		ventricular long-axis areas.
					LVEF and LVFS
Boutagy et al (52)	20/Wistar rats	Doxorubicin	2.15 mg/kg, ip	Every 3 days	Impaired systolic function and	Values calculated
	(Crl:Wl)/male		(cumulative dose	for 21 days	LV volumes and dimensions.	manually by the
			15 mg/kg)		Parameters monitored:	authors of this
					echocardiographic variables	review
					(LVEF, global longitudinal strain,
					global radial strain, LVEDV,
					LVESV, relative PWT
Lee et al (53)	150/Fischer rats/male	Doxorubicin	2.5 mg/kg, ip	Every other day	Altered LV function	Values calculated
			(cumulative dose	for 2 weeks	Parameters monitored:	manually by the
			15 mg/kg)		LVFS, LVEDd and LVESd,	authors of this
					LV end diastolic volume	review
					(LVEDV), right basal
					ventricular diastolic diameter
					(RVD1), and the RV fractional
					area change (RVFAC)
da Silva et al (54)	52/Wistar rats/female	Doxorubicin	1.25 mg/kg, ip	Three times a week	Parameters monitored:	Values calculated
				for 2 weeks	aorta-to-left atrial diameter	manually by the
					ratio, LVESd, LVEF	authors of this
						review
Mao et al (55)	160/Sprague-Dawley	Doxorubicin	2 mg/kg, ip	Once a week for	Parameters monitored:	Values provided
	rats/male			8 consecutive weeks	LVEDd, LVESd, LVPWT,	in the manuscript
					interventricular septum
					thickness (IVST), LVEF, LVFS
Deng et al (56)	42/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	6 injections	Parameters monitored:	Values calculated
	rats/male	(brand name Adriamycin)	(cumulative 15 mg/kg)	over 2 weeks	LV dimensions, LVFS, LVEF	manually by the
						authors of this
						review
Bertinchant et al (57)	45/Wistar rats/male	Doxorubicin	1.5 mg/kg, iv,	Once a week for	Parameters monitored:	Values provided
			(cumulative dose	up to 8 weeks	LVEDd, LVESd and LVFS	in the manuscript
			12 mg/kg)
Sun et al (58)	70/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Once a week for	Parameters monitored:	Values provided
	rats/male			6 consecutive weeks	LVEF, LVEDd, LVESd	in the manuscript
					and LVFS
Guerra et al (59)	12/SHR rats/male	Doxorubicin	1.5 mg/kg, ip	Once a week	Parameters monitored:	Values provided
			(cumulative dose	for 9 weeks	LVEDd, LVESd and LVEF	in the manuscript
			13.5 mg/kg)
Gao et al (60)	90/Wistar albino rats/male	Doxorubicin	2 mg/kg, ip	Every 3 days	Parameters monitored:	Values calculated
				for 30 days	The interventricular septal	manually by the
					thickness at diastole, left	authors of this
					ventricular internal diameter	review
					in diastole and systole,
					LVPWd at diastole, EF, FS
Chen et al (61)	60/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	6 injections	Parameters monitored:	Values calculated
	rats/male			over 2 weeks	LVAW, LVPWT, LVIDd	manually by the
					were measured in systole	authors of this
					and diastole.	review
					EF, FS and LV volume at
					end-systole and end-diastole
Li et al (62)	56/Sprague-Dawley	Epirubicin	8 mg/kg, ip	Every five days for	Parameters monitored:	Values calculated
	rats/male			a total of three	LV dimensions and wall	manually by the
				injections	thickness, EF, FS	authors of this
						review
Schwarz et al (8)	60/Sprague-Dawley	Doxorubicin	2.5 mg/kg, iv	Once a week	Left ventricular end-systolic	Values provided
	rats/female	(brand name Adriamycin)		for 10 weeks	and end-diastolic diameters, FS	in the manuscript
Leontyev et al (63)	46/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Once a week	LV end-systolic diameter	Values provided
	rats/male			for 9 weeks	(LVESD) and LV end-diastolic	in the manuscript
					diameter (LVEDD) + FS
Merlet et al (64)	158/Sprague-Dawley	Doxorubicin	2.5mg/kg, ip	6 injections	LV end-diastolic and -systolic	Values calculated
	rats/male		(total 15 mg/kg)	over 2 weeks	diameters (LVEDD and	manually by the
					LVESD), diastolic posterior	authors of this
					wall thicknesses (dPWth).	review
					+ LV end diastolicand systolic
					volumes (LVEDV and VESV)
					to assess LV ejection fraction
					(LVEF), whereas LV shortening
					fraction (LVSF)
Ozkanlar et al (65)	40/Sprague-Dawley	Doxorubicin	2.5 mg/kg, iv	Once a week	Left ventricular ejection	Values provided
	rats/male			for 3 weeks	fraction (LVEF) and left	in the manuscript
					ventricular fractional shortening
					(LVFS)
Hong et al (66)	12/Sprague-Dawley	Doxorubicin	5 mg/ week	Once a week	FS and ejection fraction +	Values provided
	rats/male	(brand name Adriamycin)		for 3 weeks	interventricular septal	in the manuscript
					dimension diastole; LV
					internal dimension diastole;
					LV posterior wall dimension
					diastole; interventricular
					septal dimension systole;
					LV internal dimension
					systole; LV posterior
					wall dimension systole
Teraoka et al (67)	75/Wistar rats/male	Doxorubicin	1 mg/kg, ip	15 times over a	LV diameter of the systole	Values provided
		(brand name Adriamycin)	(cumulative dose	period of 3 weeks	LVDs + LV diameter of	in the manuscript
			15 mg/kg)		the diastole LVDd.
					+ %fractional shortening
Hamed et al (68)	130/Wistar rats	Doxorubicin	Cumulative dose of	3 weeks	LV diameter	Values provided
	(Harlan)/male		15 mg/kg		in systole (LVIDs)	in the manuscript
					LVIDd, LV diameter in diastole;
					IVSd, intra ventricular septum
					in diastole
					LV posterior wall thickness
					in diastole (LVPWd)
Gabrielson et al (69)	21/Sprague-Dawley	Doxorubicin	Cumulative dose of 15	Six or three weekly	Interventricular septum	Values calculated
	rats/female		or 7.5 mg/kg	doses, respectively	diastole (IVSd) and	manually by the
					left ventricular posterior	authors of this
					wall thickness at end diastole	review
					(PWTED)
					+ LV chamber diameters were
					measured at the end of diastole
					(LVEDd) and systole
					(LVESd). EF%
Yu et al (70)	63/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Once a week	LV shortening (LVFS) was	Values provided
	rats/male			for 6 weeks	calculated as (LVEDd-	in the manuscript
					LVESd)/LVEDd 9 100,
					where LVEDd is LV
					end-diastolic diameter and
					LVESD is LV end-systolic
					diameter
					+ LV ejection fraction
Bai et al (71)	Rats	Doxorubicin	6 injections total	Within 2 weeks	LVEF; LVFS; LVEDd	Values provided
			15 mg/kg)		and LVESd	in the manuscript
Lu et al (72)	48/Sprague-Dawley	Doxorubicin	1 mg/kg on the 2nd and		LV internal end-diastolic	Values calculated
	rats/male		4th days, 2 mg/kg on the		diameter (diastolic LVID)	manually by the
			6th and 8th days,		and the posterior wall	authors of this
			3 mg/kg on the 10th		end-diastolic thickness	review
			and 12th days, and		(diastolic LVPW) + LV
			4 mg/kg on the 14th		diastolic volume (diastolic
			and 16th days, ip		LVV) and function indexes
					(stroke volume, EF and FS)
Wachtman et al (73)	30/Sprague-Dawley	Doxorubicin	2.5 mg/kg, iv	Once a week for	FS	Values provided
	rats/female			a total of 6 doses		in the manuscript
Zhang et al (74)	40/Wistar outbred rats/male	Doxorubicin	2.5 mg/kg, ip	Three times per week	The LV end-systolic diameter	Values provided
		(brand name Adriamycin)	(total 15 mg/kg)	for one week. After a	(LVSD), the LV end-diastolic	in the manuscript
				two-week interval,	diameter (LVDD), the LV
				administration for	end-systolic volume (LVSV)
				another week. These	and the LV end-diastolic
				steps were conducted	volume (LVDV) + The LV
				6 times	ejection fraction (LVEF)
					and the LV shortening
					fraction (LVFS)
Chen et al (75)	39/ Wister rats/male	Doxorubicin	2.5 mg/kg, ip	Six times for 2 weeks	LV end diastolic diameter	Values provided
					(LVEDd), LV end systolic	in the manuscript
					diameter (LVESd) and ejection
					fraction (EF) + FS + LV
					systolic pressure (LVSP), LV
					end diastolic pressure (LVEDP),
					LV maximum dP/dt and
					LV minimum dP/dt
Ha et al (76)	60/Wistar rats/male	Doxorubicin	2 mg/kg, iv	Once a week for 2, 4,	LV performance	Values calculated
		(brand name Adriamycin)		6 or 8 weeks,	LV dimensions (end-diastolic	manually by the
				consecutively	and end-systolic diameter)	authors of this
					+ EF	review
Emanuelo et al (77)	40/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Every second day	LV systolic pressure (LVSP)	Values calculated
	rats/male		(total 15 mg/kg)	for a period of	Diastolic and systolic	manually by the
				2 weeks	LV wall thickness, LVEDD,	authors of this
					and LVESD were measured	review
					+ percent LV FS
Lim (78)	52/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Six times	LVES dimensions, LVED	Values provided
	rats/male			over 2 weeks	dimensions, LVFS	in the manuscript
Hydock et al (79)	147/Sprague-Dawley	Doxorubicin	10 mg/kg, ip	Acute administration	SWT during systole (SWs)	Values calculated
	rats/male			(bolus injection)	and diastole (SWd), PWT and	manually by the
					PWT during diastole (PWd),	authors of this
					LVEDd, LVESd, FS	review
Xiang et al (80)	37/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Once a week	LVEDd and LVESd +	Values provided
	rats/male			for 6 weeks	LV FS (%)	in the manuscript
Kenk et al (81)	94/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	6 injections	LV internal diameter	Values provided
	rats/male	(brand name Adriamycin)	(total 15 mg/kg)	over 2 weeks	(LV diastolic and systolic	in the manuscript
					dimensions; LVDD and
					LVSD), LV posterior
					wall (LVPW), and intra-
					ventricular septum (IVS)
					thickness at end-diastole
					and peak systole.
					→LV volume in diastole
					and systole (LVDV, LVSV),
					stroke volume (SV),
					EF, FS, and LV mass
Katona et al (82)	23/Adult Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	Three times a week	Parameters monitored:	Values provided
		(brand name Adriamycin)		for 2 weeks	LVDDd and LVSDd,	in the manuscript
					FS, LAD, AOD
Hydock et al (83)	49/Sprague-Dawley	Doxorubicin	1.5 mg/kg i.p of	Once a day for	Septal wall thickness at systole	Values provided
	rats/female		(cumulative 15 mg/kg)	10 consecutive days	(SWs) and diastole (SWd),	in the manuscript
					posterior wall thickness at
					systole (PWs) and diastole
					(PWd), LVDs and LVDd,
					and FS
Hou et al (84)	40/Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	6 times for 2 weeks	LV dimensions	Values provided
		(brand name Adriamycin)			[end-diastolic diameter	in the manuscript
					(LVDd) and end systolic
					diameter (LVDs)] + % FS
					of the LV
Hydock et al (85)	74/Sprague-Dawley	Doxorubicin	1 mg/kg, ip	Once a day for	Septal wall thickness	Values provided
	rats/male		(total 10 mg/kg)	10 consecutive days	at systole (SWs) and diastole	in the manuscript
					(SWd), posterior wall thickness
					at systole (PWs) and diastole
					(PWd), LVDs and LVDd.
					+ FS, LV mass and relative
					wall thickness (RWT).
Koh et al (86)	33/Wistar rats/male	Doxorubicin	2 mg/kg, iv	Once a week	LV dimensions (the LVDd,	Values provided
		(brand name Adriamycin)		for 8 weeks	LVDs, the intraventricular	in the manuscript
					septal thickness, and the LV
					posterior wall thickness) +
					% FS of LV atrial natriuretic
					peptide; brain natriuretic peptide
Carresi et al (87)	40/Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	6 times for 2 weeks	LVESd; LVEDd; IVSs;	Values provided
					IVSd, LVPWs and LVPWd;	in the manuscript
					EF; FS
Ma et al (88)	190/Wistar rats/male	Doxorubicin	2.5 mg/kg, ip	6 times for 2 weeks	LVEDD) and LVESD	Values provided
					+ FS + EF	in the manuscript
Zhang et al (89)	26/Sprague-Dawley	Doxorubicin	4 mg/kg, ip	Twice per week	Diastolic interventricular	Values calculated
	rats/male		(cumulative dose	for 2 weeks	septum thickness (IVSTd),	manually by the
			16 mg/kg)		systolic interventricular septum	authors of this
					thickness (IVSTs), + EF + FS	review
Sun et al (90)	32/Sprague-Dawley	Doxorubicin	20 mg/kg, ip	Acute administration	(LVEF) from EDV and ESV,	Values provided
	rats/male		5.0 mg/kg, iv	(single dose)	+ EDV and ESV	in the manuscript
					+ LVFS
Zhu et al (91)	50/Adult Sprague-Dawley	Doxorubicin	2 mg/kg/week	6 weeks	Ejection fraction	Values provided
	rats/male					in the manuscript
Croteau et al (92)	12/ Fisher rats/male	Doxorubicin	2 mg/kg, iv	Once a week	Left ventricular function	Values provided
				for 6 weeks	Left ventricle ejection fraction	in the manuscript
Ikegami et al (93)	14/Sprague-Dawley/NM	Doxorubicin	2.5 mg/kg, ip	3 times a week	LVDd and LVFS + FS	Values provided
				for 2 to 6 weeks		in the manuscript
Hiona et al (94)	24/Sprague Dawley	Doxorubicin	Cumulative dose of	Once a week	LVFS	Values provided
	rats/female		25 mg/kg, ip	for 6 weeks		in the manuscript
Tang et al (95)	40/Sprague-Dawley	Doxorubicin	2.5 mg/kg, ip	Once a day for	Parameters monitored:	Values provided
	rats/male			a total of 6 times	LVEF, LVIDd, LVIDs,	in the manuscript
					LVPWd, LVPWs, left ventricle
					% EF, and left ventricle % FS
Migrino et al (96)	31/Sprague Dawley	Doxorubicin	2.5 mg/kg, iv	Once a week	FS monitored	Values provided
	rats/male			for 10 or 12 weeks		in the manuscript
Liu et al (97)	24/Sprague-Dawley	Doxorubicin	Each dose consisted of	At 1st, 3rd, 5th, 7th,	Parameters monitored:	Values provided
	rats/male	(brand name Adriamycin)	1, 1, 2, 2, 3 and 3 mg/kg,	9th and 11th day,	interventricular septum	in the manuscript
			ip (cumulative dose	respectively	thickness of systolic, IVSd,
			12 mg/kg)		LVIDd, LVISd, LVPW,
					LVPWd, EF, FS
Liu et al (98)	120/Sprague Dawley	Doxorubicin	3.3 mg/kg, iv	Once a week		Values provided
	rats/NM			for 4 weeks		in the manuscript

LV, left ventricular; LVEF, LV ejection fraction; LVFS: LV fractional shortening; BNP, brain natriuretic peptide; PWT, posterior wall thickness; AWT, anterior wall thickness; SWT, septal wall thickness; BP, blood pressure; HR, heart rate; LVSP, LV systolic pressure; LVDP, LV diastolic pressure; LVEDd, LV end-diastolic diameter; LVESd, LV end-systolic diameter; LVEDV, LV end-diastolic volume; LVIDd, LV internal diastolic diameter LVISd, LV internal systolic diameter; LVPWs, LV systolic wall thickness; LVPWd, LV diastolic wall thickness; IVSd, intraventricular septum in diastole; LAD, left atrial diameter; AOD, aortic diameter; ip, intraperitoneally; iv, intravenously; NM, not mentioned; SD, Sprague-Dawley.

In Figs. 2–5, the normal and suppressed values of the two main echocardiographic indices discussed, %EF and %FS, respectively, are presented. Reported baseline (normal) %EF values in rats vary (55-96.5%). In 78.2% of the studies reviewed, normal values range from 70 to 90%. High %EF values (>90%) are reported in 14% of the studies. In contrast, normal %FS values present even higher variability (25-84.2%). The majority (66.7%) of the values, though, are reported to be within the range of 40 and 60%.

Figure 2.

Normal (baseline) LVEF values in rats before anthracycline administration as reported in 57 relevant studies reviewed in the present report. LVEF, left ventricular ejection fraction.

Figure 5.

Suppressed LVFS values in rats due to anthracycline toxicity as reported in 78 relevant studies reviewed in the present report. LVFS, left ventricular fractional shortening.

Exposure to anthracyclines suppresses both echocardiographic indices. In the 86 studies reviewed in the present report, Doxorubicin is almost universally used to induce cardiotoxicity, along with Daunorubicin and Epirubicin in two studies (Table I). The structures of the three anthracyclines used are presented in Fig. 6. Anthracyclines were administered with order of appearance either via intraperitoneal injection, intravenous injection or orally with the feed. The doses were administered once, twice, three times per week. The duration of the dose administration spans from one week to ten weeks. In most of the experiments, the benchmark for terminating the administration was the proof of cardiac toxicity. The echocardiography values suggest that there is no specific dose regime threshold which indicates the establishment of the effect, but it is specific to each experiment and probably dependent on other factors such as age and general condition of the animals.

Figure 6.

Chemical structures of the three anthracyclines used to induce cardiotoxicity in the studies reviewed in the present report.

The suppressed %EF values reported from rats after anthracyclines administration vary from 31 to 91% (Fig. 4). EF values 50–80% are reported in 72.3% of the studies reviewed. Suppression of the %EF due to anthracycline administration varies from 10 to 40% compared to the normal values in more than two thirds of the studies reviewed (71.7%) (Fig. 7). On the other hand, suppressed %FS values ranging from 14 to 71.8%, present a more narrow distribution (%FS values 20–50% in 84.6% of the studies). As shown in Fig. 7, a more equal distribution of the %FS suppression due to anthracycline toxicity is observed with approximately one fourth of the studies reporting 20–30% and 30–40% suppression, respectively. It is evident from Figs 8 and 9 that normal and suppressed %EF and %FS values separate sufficiently well. The rat strain does not seem to influence either the normal or the suppressed %EF and %FS values (Fig. 10).

Figure 4.

Suppressed LVEF values in rats due to anthracycline toxicity as reported in 54 relevant studies reviewed in the present report. LVEF, left ventricular ejection fraction.

Figure 7.

Percentiles distribution of % suppression of LVEF and LVFS due to anthracycline toxicity as mentioned in the studies reviewed in the present report. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening.

Figure 8.

Scatter plot of normal (baseline) and suppressed LVEF values in rats due to anthracycline toxicity as reported the studies reviewed in the present report. LVEF, left ventricular ejection fraction.

Figure 9.

Scatter plot of normal (baseline) and suppressed LVFS values in rats due to anthracycline toxicity as reported in the studies reviewed in the present report. LVFS, left ventricular fractional shortening.

Figure 10.

Normal and suppressed LVEF and LVFS values for the two main rat strains used in the studies reviewed in the present report. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening.

Only 11 studies used an acute administration scheme, with 3–20 mg/kg bw anthracycline single injection either intravenously or intraperitoneally. Most of the studies used a prolonged administration period, from 2 weeks (33 studies) up to 10 weeks, and cumulative doses ranging from 1 to 20 mg/kg bw. All dosage schemes were carefully selected to induce cardiotoxicity and did not seem to affect the suppression of %EF and %FS monitored.

Discussion

Myocardial contractility suppression due to anthracycline administration is of increasing interest and represents a major challenge in the clinical setting. At the same time in a preclinical stage it serves as a model for the assessment of both new chemotherapeutic and cardioprotective agents to be introduced in clinical practice. The myocardial toxicity of anthracyclines is known to be affected by sex and age, along with a number of cardiovascular risk factors and comorbidities (99). It is found that anthracycline related congestive heart failure reaches 10% of patients older than 65 years at usual doses (100). While in early studies it was thought that EF cannot accurately predict congestive heart failure attributed to doxorubicin (100), current perspective is that anthracycline-related cardiotoxicity is manifested by a progressive continuous decline in LVEF (1) and identifying subclinical myocardial dysfunction related to anthracycline treatment has great therapeutic implications (2).

Preclinical animal studies are essential in cancer chemotherapy research along with the evaluation of the cardiotoxic propensity of the chemotherapeutic agents. The current recommendations for prevention of cardiac events from cancer chemotherapies are largely based on recommendations. The American Society of Clinical Oncology, for example, recommends active screening and prevention of modifiable cardiovascular risk factors, such as tobacco use, high blood pressure, high cholesterol, alcohol use, obesity and physical inactivity (101). A well characterized animal model for defining cardiotoxicity due to chemotherapy and the treatment thereof is of great importance for clinical practice, as it will enable physicians to base their decisions not only on epidemiology but also on observations developed using concrete data from animal studies.

In the present review, the range of the main echocardiographic indices, namely EF and FS, used in describing anthracycline cardiotoxicity in rats was summarized along with the normal values of the said indices presented in the respective studies. In the graphic representation, it seems that normal and suppressed values due to anthracyclines administration for the two echocardiographic indices are well separated. This provides the first evidence for the possibility of setting a cut-off point for defining anthracycline cardiotoxicity in rats with an in-depth future meta-analysis.

In the current study a wide range of EF and FS decline due to anthracycline administration was observed. However, the trends of the said decline are easily identified, especially for FS values, thus rendering the establishment of minimum cut off values of decline feasible. The question remains, as it has also been identified for humans, whether the absolute suppressed values of EF and FS, combined or separately, or the % suppression caused by anthracyclines should be used to describe cardiotoxicity, and which of the two approaches could be more effective in prevention. In our study, it seems that setting a range for % suppression of EF and FS could be more efficient in identifying early cardiotoxicity by counteracting the intra-individual variation of the absolute values.

In the current in depth review analysis, we did not identify differences between rat strains in terms of suppressed EF and FS values due to anthracycline administration. This is an interesting finding as it seems that the usual strains used in rat studies are equally prone to the cardiotoxic anthracycline potential. In animal models of genetically programmed hypertension and heart failure, it is found that doxorubicin administration did not lead to lower myocardial contractility compared to non-genetically modified strains (102). In addition, in the current systematic review, acute and chronic anthracyclines cardiotoxicity models were found equally potent in inducing cardiotoxicity based on evaluated echocardiographic indices.

Currently, when assessing chemicals toxicity, cardiac effects if monitored and detected in animal studies, mainly on the tissue level, are considered by the authorities, but cardiotoxicity, as such, is not described as a separate hazard class of chemical substances through the available regulations, both at a European level and world-wide. Therefore, chemicals other than pharmaceutical agents are recognised to be cardiotoxic after having exerted such deleterious effects on humans, based on epidemiological studies. In a previous review of our research team, the cardiac pathology and function impairment due to exposure to pesticides revealed that several cardiovascular complications have been reported in animal models including electrocardiogram abnormalities, myocardial infarction, impaired systolic and diastolic performance and histopathological findings, such as haemorrhage, vacuolization, signs of apoptosis and degeneration (103). In addition, there is evidence that short and/or long-term exposure to anabolic androgenic steroids is linked to a variety of cardiovascular complications which could be identified by using echocardiography or biochemical markers (10,104,105). The published data suggest clearly that there is a need to establish regulatory criteria for assessing cardiotoxicity as an inherent property of a chemical substance well in advance, and characterize the risk of exposure to such chemicals through a well-developed regulatory network based on animal models, as is the case for other human health hazard classes, such as carcinogenicity. Regulatory established criteria will enable international organizations to early identify cardiotoxic effects and classify chemicals in order to avoid long-term cardiovascular complications. Specific classification criteria should be developed based on anatomical, histopathological, echocardiographic and biochemical criteria in animals developed in a way that could exclude confounding factors in the development of the observed cardiotoxicity. The results of the present study are promising in identifying echocardiographic criteria in rats for the establishment of cardiotoxicity. Further studies and meta-analyses are needed in order to evaluate other species, commonly used in research, and explore the possibility of early recognizing the onset of cardiotoxicity, possibly through monitoring of biochemical markers based on understanding of the mode of action.