Hepatic and renal damage by alcohol and cigarette smoking in rats

Abstract

Chronic use of alcohol and tobacco cigarettes is associated to millions of deaths per year, either by direct or indirect causes. However, few studies have explored the additional risks of the combined use of these drugs. Here we assessed the effect of the combined use of alcohol and cigarette smoke on liver or kidney morphology, and on biochemical parameters in chronically treated rats. Male Wistar rats were allocated to receive 2 g/kg alcohol orally, which was followed by the inhalation of smoke from six cigarettes during 2 h (ALTB group) for 28 days. Other groups received alcohol alone (AL) or were exposed to cigarette smoke (TB) alone and were compared to control (CT) rats, which received water followed by ambient air. On day 29, rats were euthanized and blood samples were collected for aminotransferase enzymes (AST and ALT), creatinine, and urea analysis. Liver and kidney were weighted, dissected, fixed, and stained with hematoxylin and eosin for morphological analysis. Our results showed that necrosis was elevated in the AL, TB, and mainly the ALTB group in both liver and kidney of rats. Serum levels of AST and ALT were reduced by cigarette smoke exposure, independently of alcohol use. Serum creatinine levels increased after tobacco smoke exposure. On the other hand, TB and AL groups decreased serum urea levels, and their association restored that decrease. Absolute liver and kidney weights were lower in the cigarette smoke exposure rats. Lastly, body weight gain was lower in TB group and combined use restored it. Thus, we may infer that the use of alcohol, exposure to tobacco cigarette smoke or, mainly, their association promotes liver and kidney injuries, and this damage is related with biochemical changes in rats.

Introduction

Alcohol and tobacco are the most frequently abused drugs in the world. According to the World Health Organization, the abuse of alcohol is associated with over 3.3 million deaths each year [1]. The data for cigarette smokers is even more alarming, killing nearly 6 million people each year and representing about one death every 6 s [2].

Although moderate doses of alcohol have antioxidant and cardioprotective effects [3], chronic or abusive alcohol drinking are associated with toxic effects on different organs and are the major cause of liver disease [4]. Alcoholic fatty liver, alcoholic hepatitis, and cirrhosis are some of the most prevalent diseases among heavy alcoholics [4]. Hepatic steatosis is also common, affecting more than 90% of the heavy drinkers. Furthermore, alcohol and its toxic metabolite, acetaldehyde, promote cell membrane damage to the nephron, reducing the synthesis of polyunsaturated fatty acids, cholesterol, and phospholipids [5]. Indeed, alcoholism is associated with tubular dysfunction and renal failure, promoting electrolyte imbalance [5].

Tobacco smoking, on the other hand, is more frequently associated with increased morbidity and mortality from cardiovascular and respiratory causes, such as myocardial infarction, sudden death, stroke, chronic obstructive pulmonary disease, lung cancer and many other cancers [6]. However, smoking also increases the risk of liver cirrhosis independently of combined alcohol intake [7], and both active and passive smokers are more likely to develop non-alcoholic fatty liver disease [8]. Tobacco smoking also has a negative impact on renal function and is a major risk factor for renal cancer and chronic kidney diseases [9, 10]. In rodents, 6 months of daily chronic exposure to cigarette smoke increases renal fibrosis [11]. In humans, smoking increases the risk of proteinuria and promotes renal failure, mainly in hypertensive, diabetic, and elderly smokers, or those with pre-existing renal disease [12, 13].

Circulating levels of biochemical markers such as creatinine, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), also point to the conclusion that the association of alcohol and tobacco increases tissue damage and reduces the function of certain organs [14, 15]. AST and ALT are widely distributed in human tissues and increase their plasma levels after injury or infectious processes in the liver, kidneys, and other tissues. ALT is found mainly in the cytoplasm, while AST is found in the cytoplasm and also in the mitochondria [16]. For instance, after 12 weeks of alcohol intake, urine urea levels are decreased, while plasma urea, as well as serum AST and ALT levels, are increased in rats [14]. Although some studies have shown that creatinine levels are not changed by long term alcohol consumption [14, 15], the opposite has been reported [17]. On the other hand, smokers present decreased urea and creatinine plasma levels, accompanied by an increase of uric acid plasma level [18].

Hepatic and renal damage may be explained by direct or indirect effects of these two drugs, by alcohol per se or acetaldehyde, or by inhalation of the gaseous and particulate constituents in the cigarette smoke [19, 20]. Despite the known health risk of human exposure to alcohol and cigarette smoke, few studies have explored the effects of the combined use of these drugs on the liver and kidney, or on biomarkers for their function. Thus, our objective was to evaluate the hepatic and renal damage after chronic exposure to alcohol, tobacco cigarette smoke, or their combination in rats. We hypothesize that the combined exposure to alcohol and cigarette smoke is more deleterious to the liver and the kidney than the isolated use of one of them.

Material and methods

Animals

Forty male adult (~ 280 g) Wistar rats, born and reared in the Center for Reproduction and Experimentation of Laboratory Animals (CREAL-UFRGS), Porto Alegre, Brazil, were housed in polypropylene cages (33 × 17.8 × 40 cm) (n = 3 rats/cage) under controlled environmental conditions (12 h light/dark cycle: 7 am–7 pm; temperature: 22 ± 2 °C; air humidity: 55%). They had free access to water and food (Nuvilab, Colombo, Paraná, Brazil) and were weighed every 2 or 3 days for alcohol dose adjustment. All procedures were performed in accordance with national and international guidelines and were previously approved by the Ethics Committee for Animal Use at UFRGS (CEUA-UFRGS # 25022). All efforts were made to minimize the number of animals used and their suffering.

Alcoholic solution and cigarette smoke exposure

Ethanol (99%, Merck, São Paulo, Brazil) was diluted to 20% (w/v) in tap water and administered by oral gavage (2 g/kg). Alcohol-negative control groups received tap water at a volume of 10 mL/kg. Tobacco cigarettes were from a commercial brand, containing 0.8 mg of nicotine/cigarette, according to the manufacturer. They were burned in an appropriated apparatus and smoke was dragged to a hermetically sealed glass chambers (50 × 30 × 30 cm), with a controlled negative airflow ventilation (10 L/min), maintained constant by a vacuum pump [21–23] (Fig. 1). Cigarette-negative control groups were also exposed to the chamber but with environmental fresh air circulating.

Fig. 1.

Experimental procedure. Alcohol or tap water were administered before cigarette smoke or environmental air in hermetic chambers. Cigarettes were burned with a 10-min interval period between each other using a controlled negative airflow ventilation kept constant (10 L/min) by a vacuum pump. Daily treatment: 4 g/kg alcohol and 12 burned cigarettes; 4 weeks. At the end of the experiments, blood, liver, and kidney were biochemically and morphologically evaluated. Groups ⇒ CT: water + air; AL: alcohol + air; TB: water + smoke; ALTB: alcohol + smoke (n = 8–9/group)

Experiental procedures

Animals were divided (n = 10/group) in the control (CT) or tobacco cigarette smoke (TB) groups, receiving tap water, and in the alcohol (AL) or alcohol + cigarette smoke (ALTB) groups, receiving 2 g/kg alcohol orally twice a day (9 AM and 2 PM), for 28 days. Immediately after the alcohol administration, animals were placed in hermetic chambers (n = 5 rats/chamber) for 2 h, with fresh air (CT and AL groups) or tobacco cigarette smoke exposure (6 cigarttes; TB, and ALTB groups) (Fig. 1). Cigarettes were burned with a 10-min interval period between each other to avoid intoxication of rats. The total daily treatment consisted of 4 g/kg alcohol and 12 burned cigarettes. We have previously shown that, 60 min after the last exposure to cigarette smoke, plasma cotinine levels were 1006.4 ± 170.1 ng/dL while being undetectable in the control group [23]. On day 29, 18 h after the last exposure to alcohol/water and/or cigarette smoke/environmental air, rats were euthanized and their blood was collected and centrifuged, and the resulting serum was stored at − 80 °C for later determination of biochemical parameters (transaminases, urea, and creatinine). Liver and kidneys were carefully dissected, weighed, and fixed with a solution of 4% paraformaldehyde solution for subsequent histological processing. Tissue weights were normalized to body weight and presented as absolute and relative values. Weigh body changes were measured every 2–3 days for adjustment of alcohol dose and to monitor these parameters along the experiment. Body weight was evaluated on day 0 (week 0), week 1, 2, 3, and 4.

Morphological analysis

Livers and kidneys were processed according to conventional histological techniques and included in paraffin (Paraplast®, Sigma Aldrich®, São Paulo, Brazil). Tissues were then sectioned with a microtome at 4 µM, with a 50 µM interval between them, and arranged 4 slices per blade. Slides were stained with hematoxylin and eosin and morphological evaluation were observed in optical microscope with 100 × and 400 × by a trained evaluator and doubled checked by a second one, with both observers being blind for the groups. Parameters of damage were observed and quantified in 10 fields in each Sect. (60 fields per animal) by assigning scores that had been standardized from 0 to 3, being that 0: no change; 1: slight change; 2: moderate change; and 3: severe change. After the analysis, the 0 and 1 classification were added, as well as the 2 and 3 to compare the morphological parameters, using absolute frequency and relative frequency (%) (Tables 1, 2). Morphological changes in the liver were considered by the presence of hepatocyte swelling, hydropic degeneration, inflammatory cells, necrosis, perivascular infiltrate, sinusoid enlargement, and vascular congestion [24, 25]. For the kidney, the parameters were: cortical vascular congestion, convoluted distal tubule dilation,cytoplasmic vacuolization,presence of hyaline material; infiltration area; inflammatory cells, necrosis, swelling of bone marrow and of cortical cells [24, 25]. To exemplify the impairment induced by drugs, we represented these accumulated scores of damages as photomicrograph exemplification of the groups (Figs. 2, 3).

Table 1.

Absolute frequency for scores of different histological parameters in the liver of rats chronically treated with saline (CT), alcohol (AL), exposed to tobacco cigarette smoke (TB), or both drugs (ALTB)

Histological parameters	Scores								p
	CT		AL		TB		ALTB
	0–1	2–3	0–1	2–3	0–1	2–3	0–1	2–3
Hepatocyte swelling	8	0	6	2	8	0	8	0	0.094
Hydropic degeneration	8	0	6	2	8	0	6	2	0.206
Inflammatory cells	2	6	0	8	0	8	0	8	0.094
Necrosis	7	1	2	6*	1	7*	2	6*	0.008#
Perivascular infiltrates	7	1	4	4	5	3	1	7	0.024##
Sinusoids enlargement	8	0	7	1	8	0	7	1	0.545
Vascular congestion	8	0	6	2	8	0	7	1	0.257

Scores: 0 = no; 1 = mild; 2 = moderate; 3 = severe damage, n = 8–9/group; 60 sections/rat; chi-square test followed by residual analysis

*Different from scores 2–3 in the CT group (p ≤ 0.05)

^#p ≤ 0.05 in the chi-square analysis

^##Difference between groups (p ≤ 0.05) in the chi-square test, but no effect detected in the residual analysis (p > 0.05)

Table 2.

Absolute frequency for scores of different histological parameters in the kidney of rats chronically treated with saline (CT), alcohol (AL), exposed to cigarette smoke (TB) or both alcohol and smoke (ALTB)

Histological parameters	Scores								p
	CT		AL		TB		ALTB
	0–1	2–3	0–1	2–3	0–1	2–3	0–1	2–3
Convoluted distal tubule dilatation	8	0	8	0	9	0	8	1	0.413
Cortical vascular congestion	8	0	5	3	7	2	4	5	0.080
Cytoplasmic vacuolization	7	1	8	0	8	1	6	3	0.262
Hyaline material	6	2	4	4	5	4	9	0	0.092
Infiltration area	8	0	8	0	9	0	7	2	0.207
Inflammatory cells	8	0	6	2	8	1	6	3	0.284
Necrosis	7	1	3	5*	2	7*	1	8*	0.007#
Swelling bone marrow cells	8	0	8	0	9	0	9	0	0.999
Swelling cortical cells	8	0	8	0	8	0	8	1	0.999

Scores: 0 = no; 1 = mild; 2 = moderate; 3 = severe damage. n = 8–9/group; 60 sections/rat. Chi-square test followed by residual analysis

*Different from the scores 2–3 in the CT group after residual analysis (p ≤ 0.05)

^#p ≤ 0.05 in the chi-square analysis

Fig. 2.

Animal liver photomicrograph (400×). Effect of chronic administration of tap water (CT) alcohol (AL), exposure to cigarette smoke (TB) or their association (ALTB) on the liver tissue. Staining: hematoxylin–eosin. Increase (400×). a CT group, showing no histological changes; b AL group, showing areas of necrosis (→), hepatocyte swelling (⇒) and B1:lipid vacuoles, occupying most of the cytoplasm of hepatocytes, pushing the nucleus and other organelles to the cell periphery, suggestive of hepatic dysfunction steatosis ( ; green arrows); c TB group, showing areas of necrosis (→); d ALTB group, showing areas of necrosis (→), in a state of swollen cells (⇒) and perivascular inflammatory infiltrates (→)

Fig. 3.

Animal kidney photomicrograph (400×). Effect of chronic administration of tap water (CT), alcohol (AL), exposure to cigarette smoke (TBC) or their association (ALTB) on morphological parameters of renal tissue. Staining: hematoxylin–eosin (400×). a CT group, showing no histological changes; b AL group, showing areas of necrosis (→); c TB group, showing areas of necrosis (→); d ALTB group, showing areas of necrosis (→) and cytoplasm vacuolization ( green arrow)

Assessment of biochemical parameters

Serum concentrations of AST, ALT, creatinine, and urea were determined by colorimetric or enzymatic method in an automatized apparatus (CT 600 I, Labimbráz, Buenos Aires, Argentina), using commercial kits (Wiener, Buenos Aires, Argentina). Values were expressed as mg/dL.

Statistical analysis

Biochemical parameters and the weight of organs were evaluated by a one-way analysis of variance (ANOVA), followed by a Tukey test to detect differences between groups. Morphological parameter scores, which were quantified as absolute frequency, were analyzed by the chi-square test, followed by residual analysis when necessary. For the body weight observed along four weeks, a two-way repeated measures ANOVA was applied, followed by the Tukey test when necessary. Lastly, a Pearson's correlation test was used to verify associations between quantitative parameters (Sigma Stat®; Jandel Scientific Co., San Jose, CA—USA). Statistical significances were considered when p ≤ 0.05.

Results

Under our experimental conditions, we detected morphological changes in the liver and kidney of rats according to the drug exposure. In the liver (Table 1, Fig. 2), there was an increase in the proportion of moderate or severe damage (2 or 3 score) for the necrosis parameter of all intervention groups when compared to controls [chi-square: p = 0.008; CT: 7/1 (87.5%/12.5%); AL: 2/6 (25%/75%); TB: 1/7 (12.5%/87.5%); and ALTB groups: 2/6 (25%/75%), respectively for 0–1/2–3 scores and percentage of damage; Table 1]. Despite a statistically significant difference in the chi-square analysis for the perivascular infiltrate parameter (p = 0.024), in the residual analysis it was not possible to identify which group was different from each other [chi-square: p > 0.05; CT = (7/1) 87.5%/12.5%; AL: (4/4) 50%/50%; TB: (5/3) 62.5%/37.5%; and ALTB groups (1/7) 12.5%/87.5%, respectively for 0–1/2–3 scores and percentage of damage]. Nevertheless, we may infer that the damage was related to the association between alcohol and smoke cigarette, since the ALTB group showed 7 times higher moderate or severe damages than the CT group. Figure 2 shows some morphological changes in the liver of rats, with no apparent necrosis in the CT group (Fig. 2a). However, the AL (Fig. 2b), TB (Fig. 2c), and ALTB (Fig. 2d) groups show typical necrosis. Moreover, it is possible to identify hepatocyte swelling in the AL and ALTB groups, as well as perivascular inflammatory infiltrate in the ALTB group, even though there was a lack of statistical significance for these parameters. We also found a steatosis profile in some samples from the AL group (Fig. 2b1), indicating that our experimental conditions were able to evoke liver toxicity.

In the kidney, we observed almost the same pattern of damage presented in the liver (Table 2, Fig. 3). Necrosis scores were higher in AL, TB, and ALTB than CT rats [chi-square: p = 0.007; CT = 7/1 (87.5%/12.5%); AL: 3/5 (37.5%/62.5%); TB: 2/7 (12.5%/87.5%); and ALTB groups: 1/8 (22.5%/77.5%), respectively for 0–1/2–3 scores and percentage of damage; Table 2]. Figure 3 shows some morphological changes in the kidney of rats, with apparent severe damage in AL, TB, and ALTB groups. Some cytoplasmic vacuolization in the ALTB group was also observed, although it was not statistically different from other groups (Fig. 3d). Moreover, despite the lack of statistical significance, cortical vascular congestion was noticeable in the three treated groups, whit AL and TB groups having an intermediate damage frequency (37.5% and 25%, respectively) and ALTB group suggesting an additive damage effect (62.5%).

The analysis of biochemical parameters showed a similar pattern for AST and ALT, decreasing their concentration in the plasma only in the smoke cigarette exposed animals (TB and ALTB) compared to the environmental air exposed groups (CT and AL), independently of alcohol treatment (AST: p = 0.020, Fig. 4a; ALT: p = 0.030, Fig. 4b). Additionally, urea levels were lower in TB (p = 0.009) and AL (p = 0.049) groups than the CT group (Fig. 4c). Although the association of alcohol and cigarette smoke appears to prevent a decrease in urea levels, ALTB group was only statistically different from the AL group (p = 0.049), and not against the TB (p = 0.211) or CT groups (p = 0.855). The other biochemical marker evaluated, creatinine, showed that tobacco exposure (TB and ALTB groups) increased the creatinine plasma levels compared to animals that were exposed to environmental air (CT and AL groups), independently to the alcohol exposure (p = 0.010; Fig. 4D).

Fig. 4.

Chronic administration of tap water (CT), alcohol (AL), exposure to cigarette smoke (TB) or their association (ALTB) on the plasma levels of (a) AST, (b) ALT, (c) urea, and (d) creatinine (b), AST (c). ANOVA 2 W-RM followed by Tukey test. *Different from air environmental exposure; ^#Different from CT group; ^%Different from ALTB group

Regarding tissue weight, the two-way ANOVA showed that alcohol and cigarette smoke factors influenced the absolute liver weight of animals by interacting with each other (p_interaction = 0.047). The residual analysis showed that TB group presented lower liver weight than the CT (p = 0.024) and ALTB (p = 0.022) groups, and the association with alcohol (ALTB) reversed the cigarette smoke-induced decrease (Table 3). For the absolute kidney weight, we found a difference in the tobacco cigarette smoke versus environmental air factor (p = 0.027). The rats exposed to the cigarette smoke (TB and ALTB groups) have lower kidney weight compared to the environmental air-exposed animals (CT and AL groups) (Table 3). However, when we corrected the liver and kidney weight according to the body weight all differences disappeared (p > 0.05).

Table 3.

Absolute and relative liver and kidney weight of rats after 28 days of treatment with tap water (CT), alcohol (AL, 4 g/kg/day, orally), cigarette smoke exposure (TB, 12 cigarettes/dy, inhalation), or their combination (ALTB)

Group	Liver				Kidney
	Absolute (g)	p	Relative (%)	p	Absolute (g)	p	Relative (%)	p
CT	11.3 ± 0.3	TBxNTB0.172 ALxNAL0.185 INT0.047#	3.18 ± 0.08	TBxNTB0.738 ALxNAL0.203 INT0.314	2.53 ± 0.08	TBxNTB0.027* ALxNAL0.270 INT0.242	0.71 ± 0.02	TBxNTB0.354 ALxNAL0.180 INT0.353
AL	11.0 ± 0.7		3.20 ± 0.10		2.44 ± 0.06		0.71 ± 0.02
TB	9.8 ± 0.3&		3.10 ± 0.05		2.35 ± 0.03		0.75 ± 0.01
ALTB	11.2 ± 0.2		3.34 ± 0.06		2.36 ± 0.04		0.71 ± 0.02

Two-way ANOVA followed by the Tukey test; mean ± SEM (n = 9–10/group). p_TBxNTB values represent the factors of tobacco smoke versus environmental air; p_ALxNAL values represent the factor of alcohol versus tap water; p_INT represents the interaction between the two factors

*Cigarette smoke exposure (TB + ALTB) is different from environmental air exposure (CT + AL)

^#Interaction between factors

^&Different from CT (p = 0.024) and ALTB (p= 0.022) groups. No statistical significance was found in the ANOVA analysis of relative weight (p > 0.05)

The last parameter observed was the total body weight during the four treatment weeks (Fig. 5). Cigarette smoke exposure rats (TB group) decreased their body weight compared to the CT group in the third and fourth weeks (week 3: p = 0.008; week 4: p = 0.001), and in the second week when compared to the AL group (p = 0.024). Alcohol treatment alone was not able to change body weight compared to the CT group (p > 0.05). However, the association of alcohol and cigarette smoke (ALTB group) decreased body weight only against the AL group in the third week (p = 0.024). Taking all this information together, it is possible to observe that, by the end of the experiment, TB group gained less weight than CT and AL groups, while the ALTB group remained with an intermediate profile.

Fig. 5.

Chronic administration of tap water (CT), alcohol (AL), exposure to cigarette smoke (TB) or their association (ALTB) on body weight of the rats. ANOVA 2 W-RM: interaction between factors group and week with p < 0.001. ^#Different from AL group (p < 0.05); *Different from the CT group (p < 0.05)

Lastly, the correlation test did not show any association between the morphological and biochemical parameters, or even between themselves, except to the predictable direct association between AST and ALT (r = 0.474; p = 0.006).

Discussion

Our results show that the use of alcohol or tobacco cigarette or, to the greatest extent, the combination of both, induced damage in the liver and kidney of rats. They promoted necrosis in both organs, which is the most important parameter in the morphological analysis and can be identified by the absence of cellular connections and consequent disruption of the tissue, as well as cell loss and decreased staining boundary between nucleus and cytoplasm [25]. Other parameters support the hypothesis that the combination of both alcohol and cigarette elicit additional damage, with visually evident changes in the morphological analysis of samples. This indicates that all the injuries promoted by the drugs lead to alterations detected not only by necrosis, but also by some secondary cell damage outcomes. Though classified here as necrosis, the destruction of cells is also related to the apoptosis mechanism, which is frequently associated with normal cellular regeneration and control liver tissue growth [26]. Studies show that animals displayed a marked injury with liver cell apoptosis after high and chronicle alcohol doses [27, 28], with the participation of several components, such as oxidative stress, iron, cytochrome P450 2E1, among other factors [29].

In the present study, necrosis in the AL, TB, and ALTB groups was more prevalent than in the CT group, evidencing both liver and kidney toxicity for both drugs. Hepatic necrosis is also observed in several animal studies after alcohol administration, from chronic ethanol treatment to a single dose protocol [30–32]. It is relevant to point out that almost 90% of ethanol is metabolized by oxidation in the liver [33], with some hepatotoxic effects of alcohol caused, for instance, by the dysregulation of methionine metabolism [34].

Concerning cigarette smoke toxicity nicotine administration (10 days, s.c.) causes hepatic necrosis in mice, increasing the damage caused by the co-administration of other toxic substance [35]. Furthermore, the association of nicotine (s.c.) with alcohol increases the frequency of necrosis and promote fatty liver in mice after 3 weeks of treatment [36]. Nicotine that is present in cigarette smoke also induces changes that include cytoplasmic vacuolization, interstitial cell edema, and necrosis in the pancreas of rats [37]. Although we and others have explored the effect of the association between alcohol and cigarette smoke on oxidative stress and cell proliferation, this is the first study that shows the toxic effect of this association in the liver and kidney, exploring morphological and biochemical parameters.

Additionally, we showed a significant increase in the perivascular infiltrate in the liver of rats after alcohol use or cigarette smoke exposure. Despite the lack of statistical significance in the residual analysis, the higher scores were more evident in the ALTB group. Other studies also found that the livers of animals exposed to the combined use of alcohol and tobacco cigarette smoke display perivascular infiltrate, suggesting a premalignant state [38, 39]. Indeed, Wiśniewska et al. correlated the perivascular infiltrate with an increased cell proliferation in the liver of alcohol addicted rats, suggesting an elevated vulnerability to the harmful effects of other toxic substances, such as tobacco cigarette, when they are co-administered [39]. In that study, the authors conclude that animals exposed to alcohol (9 weeks), and to cigarette smoke (5 days) plus alcohol, were characterized by higher percentage of cell impairment markers compared to the animals exposed to cigarette smoke only, though results were quite different depending on how long after the last alcohol administration the animals were euthanized (5 or 24 h) [39]. Taking in account that in the present study the alcohol intake and cigarette smoke exposure occurred during a 28-days protocol, it could be expected that the three groups may present similar liver cell pattern of damage. That tissue injury hypothesis was observed in the AL, TB, and ALTB groups compared to the CT group, with some increment in the ALTB group.

Although we have not explored inflammatory and oxidative stress parameters in the present experiment, a recent study from our group showed that the association between alcohol and cigarette smoke significantly increases pro-inflammatory and oxidative parameters in different brain areas of rats (hippocampus, striatum, and frontal cortex), when compared to alcohol or cigarette smoke alone [40]. Moreover, the brain-derived neurotrophic factor, one of the proteins responsible of brain cell regeneration, were lower in ALTB group than AL and TB groups in all brain areas studied [40]. In a previous study from our group it was also showed that alcohol and cigarette smoke co-administration decreased by 60% the cell proliferation (BrdU-labeled cells) in the hippocampus of rats, while alcohol treatment decreased labeled cells only by 40%, and cigarette smoke exposure by 26% [23]. Although all these results are from brain samples, we suggest that the combined use of alcohol and cigarette smoke also produces important damages in peripheral tissues, as some results already mentioned points to that direction.

Concerning the kidney tissue, we detected a similar pattern to what was observed in the liver, where the AL, TB, and ALTB groups significantly increased the tissue necrosis. Other parameter that also presented a different profile when compared to the CT group was the cortical vascular congestion. We noticed that the highest scores were reached by three, two, and five animals in the AL, TB, and ALT groups, respectively, whereas the CT group had none. One may suggest that the association between alcohol and cigarette smoke was almost twice more deleterious than the alcohol or tobacco smoke alone for the kidney of rats. Studies also show elevated lipid damage in the kidney of rats treated with the association between alcohol and cigarette or nicotine [41, 42].

Hepatocellular diseases generally increase plasma ALT and AST [43], and chronic alcohol intake (4 weeks) has induced this effect in rats with a daily dose 50% higher than the one used in the present protocol (6 g/kg/day) [44], as well as in a free-consumption alcohol (10%) protocol for 12 weeks [14]. Curiously, we observed a significant reduction in the serum ALT and AST levels in the TB and ALTB groups. According to some authors, the decrease in ALT and AST levels have no clinical significance, except in cases of severe cellular degeneration and/or chronic cases, when there is a loss of cellular function [45, 46]. Even though we found an elevated score for necrosis in the liver of AL, TB, and ALTB groups, and a tendency to increase scores for perivascular infiltrate in the ALTB group, we did not find a correlation between these parameters and AST or ALT levels in our rats (data not shown). Thus, we cannot infer that lower AST and ALT levels in the TB and ALTB groups represent a protective effect of tobacco cigarette smoke constituents. In fact, a different study has also shown that cigarette smoke exposure decreases cell damage in the liver of alcohol-addict rats, suggesting a protective effect [39].

We also found that plasma creatinine was significantly increased in animals exposed to cigarette smoke. Urea levels also changed but followed a peculiar pattern where the AL and TB groups decreased its levels and the association between alcohol and cigarette smoke restored them to the CT group levels. In rodents, serum urea levels are not a sensitive predictor of kidney damage, while creatinine is more sensitive and specific to assess kidney dysfunction [46]. Das and colleagues also found elevated plasma urea and creatinine levels after 12 but not 4 weeks of alcohol administration (1.6 g/kg), and these changes were associated with kidney cell necrosis [17]. Nevertheless, another study did not find any changes in creatinine or urea blood levels after alcohol use in rats (8 weeks, 20% of free intake), despite finding an increase in tubular necrosis [47]. Other study only found increased plasma urea levels without any changes in creatinine levels (12 weeks, 10% of free alcohol intake) [14]. On a clinical setting, creatinine is also unaltered in the serum of alcoholic patients, although uric acid levels are elevated [15]. Thus, one may infer that duration of alcohol consumption, as well as alcohol dosage, are preponderant on biomarkers of kidney damage. Our results of creatinine blood levels are associated with morphologically inferred organ impairment, suggesting a reduction in glomerular filtration rate and indicating renal damage in both tobacco cigarette smoke exposed groups (TB and ALTB). Additionally, these findings corroborate clinical results, which have shown that smoking induces renal damage by increasing intraglomerular and blood pressure, leading to renal dysfunction of endothelial cells in the long term [12]. Because the renal dysfunction induced by smoking is closely related to the exposure period and the number of cigarettes smoked per day [48], it implies that our model adequately mimics the human cigarette consumption pattern.

When changes in body weight throughout our study were analyzed, we found that the AL group presented a weight gain profile similar to the CT group, and both tobacco groups (TB and ALTB groups) presented a profile quite different, maintaining the body weight until the second week, with a small weight gain for the following two weeks. Indeed, the TB group presented an even smaller weight gain during the four experimental weeks, ending the experiment with almost the same weight from the first day. The same profile was found after a 4-week protocol of alcohol and/or tobacco cigarette smoke consumption, with the addition of an inverted pattern in food consumption, since alcohol is a caloric drink [49]. Moreover, even after 2 weeks of tobacco smoke exposure, these animals presented a weight loss, which persisted until week 13 [50]. Regarding the absolute weight of organs, cigarette smoke-exposed animals showed a lower liver and kidney weight, though when organs were relativized to body weight, no statistically significant differences between groups remained. Absolute liver and kidney weight were also lower in animals exposed to alcohol or other toxic substances present in cigarette smoke such as cadmium when compared to control groups, without changes on relative liver and kidney weight [14, 51].

In summary, we can infer from our results that the use of alcohol, exposure to cigarette smoke or, mainly, their association, promote liver and kidney necrosis, and that this damage is related with biochemical changes in rats. Because of the implication of these two drugs of abuse with increased risk of morbidity and mortality and the resulting elevated health costs, the low number of studies evaluating the toxic effects of this association is surprising. Being legal drugs that are often used in combination, the literature needs more studies exploring the potentiation of damage in different tissues by the combined use of these substances, both in similar and in different experimental protocols.

Funding

This study was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the Pró-Reitoria de Pesquisa UFRGS.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. This study was approved by the Ethics Committee for Animal Use at UFRGS (CEUA-UFRGS # 25022). All efforts were made to minimize the number of animals used and their suffering.