Histopathological Changes of Rat Kidney with Exposure to Chronic Thinner Inhalation

Abstract

Introduction: Organic solvents are liquid substances commonly used in everyday life at home and in industrial workplaces. These solvents are found primarily in paint as thinner. Because other narcotics are hard to find, thinner is used as a narcotic especially among youngsters of low socioeconomic level. The aim of this study is to determine the histopathological changes of rat kidney with exposure to chronic thinner inhalation. Methods: Randomized trial – the study was conducted at the animal care facility of Haydarpasa Numune Education and Research Hospital. Forty albino Wistar male rats were used throughout the experiment. Three groups of rats inhaled thinner in a glass cage for 1, 3, and 5 weeks, respectively. Ten rats inhaled only the air in the room as the control group. Results: We observed the development of inflammation in the kidneys that became more remarkable as exposure time extended. Development of inflammation because of thinner apparently differed between the groups of week 1 and week 5. There was no difference in granuloma development. If the experiment lasted longer, there might have been granuloma development. Conclusions: Based on histopathological evaluations, it was shown that exposure to chronic thinner inhalation causes detectable damage on the kidney, which becomes more prominent as exposure period extends. As we established disorders in movement and consciousness in the rats during thinner inhalation, we can conclude that thinner also has a damaging effect upon the central nervous system.

• thinner
• rat
• kidney
• inhalation

INTRODUCTION

Organic solvents are liquid substances commonly used in everyday life both in our houses and in many industrial workplaces. These solvents are found mainly in dyes as diluents, in polishes, adhesives, and dry cleaning solvents.^1,2

Cellulosic thinner is a mixture of solvents, comprising hydrocarbons, esters, glycol esters, ketones, and alcohol, and is used to decrease the viscosity of all sorts of nitrocellulose-based paints and varnishes to provide ease of use.³

People are exposed to volatile solvents by way of inhalation, through skin contact, or orally. Many are exposed to inhalation of thinner in industrial areas. But because other narcotics are hard to find and if so are expensive, thinner is used also as a narcotic especially among youngsters of low socioeconomic level and who have family issues. We encounter abuse of thinner and similar substances mostly among youngster below 20 years of age.^1,4,5

Intoxication due to volatile substance inhalation presents itself at first with euphoria, feeling energetic, and disinhibition; this is followed by relaxation, dizziness, visual and verbal hallucinations, fatigue, and somnolence. In addition, coughing, sneezing, increase in salivation, skin erythema, nausea, vomiting, photophobia, disorientation, diplopia, ataxia, speech disorders, decrease in reflexes and nystagmus can be seen during use. Chronic use causes permanent damage in the central nervous system, heart, liver and lungs, and kidneys.^1,6–11

Abuse of thinner has become a growing health issue in our country too.¹² This study was therefore conducted to determine the histological changes of kidney with exposure to chronic thinner inhalation.

MATERIALS AND METHODS

In this study, the histological changes of thinner inhalation on kidney were examined in an animal model. Forty albino Wistar male rats weighing between 110 and 160 g were used in the study. Approval was obtained from the Animal Ethics Committee of Haydarpasa Numune Education and Research Hospital. All rats were fed with standard diet and water. Except during thinner inhalation periods, the rats were kept in an environment at 22 ± 2°C, 12 hours in dark and 12 hours in light. For thinner inhalation, a closed glass cage, with a size of 50 × 34 × 20 cm was used. A cellulosic thinner used in industry, containing 63% toluene, 13% acetone, 10% isobutyl acetate, 7.5% isobutanol, 6.5% butyl glycol, was chosen for the study. It was decided to expose the rats to thinner for 20 minutes until the standing up reflex was lost.

Rats were divided into four groups according to their thinner inhalation periods: Group 1 [1 week (n = 5)]; Group 2 [3 week (n = 9)]; Group 3 [5 week (n = 10)]; Control group (n = 10). This process was carried on every day for 5 weeks, twice daily at 09:00 am and 03:00 pm. At the end of their inhalation periods, rats were anesthetized with ketamine HCl injected intraperitoneally (100 mg/kg for operation and 200 mg/kg for euthanasia) in accordance with the guidelines, and they were sacrificed according to weeks (1st, 3rd, and 5th weeks) and the control group at the end of the 5th week; then the kidneys were dissected.

Tissue specimens from the kidneys were fixed with buffered formaldehyde. Sections of 5 μm thickness were prepared from the paraffin blocks provided after routine follow-up. All the sections were stained with hematoxylin eosin. Periodic acid shift (PAS), PAS diastase, Masson's trichrome, reticulin, and silver methenamine stains were applied to the kidney sections. Microscopic slides were examined under Olympus B × 50 (Olympus Co Ltd., Tokyo, Japan) light microscope at magnifications of 4, 10, and 40.

Slides of all the groups were examined in a double-blind way. In the pathologic examination of rats that are exposed to thinner for defined periods, only the parameters of inflammation and development of granuloma were evaluated. Development of granuloma was evaluated as present or absent. And development of inflammation in the kidney was classified as absent, focal, or diffuse.

Parameters were graded according to Knodell's modified histologic activity index. The statistical analysis of this study was done with GraphPad Prisma V.3 packet program. The qualitative data were evaluated with χ²-test.

RESULTS

A preliminary study group comprising four rats was generated. Five milliliters of thinner was put into a container placed in a glass cage at room temperature, and the rats were exposed to the thinner vapor. At the beginning of thinner inhalation, it was observed that the rats gathered at the farthest possible corner away from the thinner container in the cage. After the first 5 minutes, an increase in their movement, tendency to stand up on their hind legs and deeper and accelerated respiration was observed. Between 10 and 15 minutes, difficulty in walking was observed, and after 15 minutes, it was observed that they could hardly stand up, that they tried to do it by holding the cage walls, and that they fell over each other. Around 20 minutes, it was seen that they lost their standing up reflex, they gave no response to external stimuli and they lied still. Two of the rats were taken out of the cage. The ones left inside died after 5 minutes, and it was observed that the ones left outside returned to normal after approximately 30 minutes and drank lots of water.

During this experimental study beginning at the first week, fur loss and hemorrhage around the nostrils were noticed. An increase in respiration rate and defecation amount was observed during the experiment. An increase was observed in aggressive behaviors even at periods during which they were not exposed to thinner inhalation.

There was statistically significant difference between the experiment groups regarding the development of inflammation in kidney due to thinner inhalation (χ² = 10.62, p < 0.05). This difference was observed to be between the weeks 1 and 5. In terms of granuloma development in rat kidney due to thinner inhalation (Figure 1), there was no statistically significant difference (χ² = 1.35, p > 0.05) (Table 1).

Figure 1. Rat kidney at the thinner inhalation group (granuloma formation was shown by small arrow and interstitial infiltration by large arrow).

Table 1. Summary of histopathological findings

	Control	Week 1	Week 2	Week 5
Granuloma
Absent	1	0	1	2	χ^2 = 1.35
Present	9	5	8	8	p > 0.05
Inflammation
Absent	3	4	2	1
Focal	6	0	5	5	χ^2 = 10.62
Diffuse	1	1	2	4	p < 0.05

DISCUSSION

Industrial thinner is composed of 63% toluene which is the reason for side effects.¹ The Occupational Safety and Health Administration recommends an exposure limit of 150 ppm (averaged over a normal 8-hour workday) to ensure that workers can be exposed without adverse effects. When toluene concentration exceeds 2000 ppm, there is risk of death.¹³ Repeated inhalation of toxic concentrations of thinner induces a wide range of responses in kidney, depending on concentration and duration of exposure. There are studies reporting hepatic, renal, cardiovascular, and hemopoietic system damages in thinner addicts.^1,6,14,15

In a study conducted in 2000, two types of rats were exposed to inhalation of 2-butoxyethanol chemical which is also present in thinner, for defined periods every day for 14 weeks and 2 years, respectively. At the end of the period in the kidneys of the sacrificed subjects, renal tubular degeneration and increase in hemosiderin pigmentation were detected.¹⁴

Batlle et al. evaluated renal tubular acidosis developed in five addicts smelling toluene. In some of them, besides renal tubular acidosis, aminoaciduria, hypophosphoremia, and hypocalcemia had developed.¹⁵

In a case report by Erramouspe et al., it was established that the infant of a mother who has smelled dye thinner containing toluene during her pregnancy was born with renal tubular acidosis.¹⁶ The reason for renal toxicity due to smelling of adhesives seems to be toluene. Because of abuse, distal renal tubular acidosis, metabolic acidosis with high anion gap, Fanconi syndrome, urolithiasis, glomerulonephritis, and Goodpasture syndrome can develop. In patient smelling adherents who have mesangiocapillary glomerulonephritis, immune complex accumulation in the kidneys has been reported. In people smelling toluene chronically hematuria, sterile pyuria, and proteinuria have been defined. Distal renal tubular acidosis developing due to toluene can revert as the smelling dependancy is given up. Eighty percent of the smelled toluene is metabolized to benzoic and hippuric acid by hepatic cytochrome P450 system and alcohol dehydrogenase enzyme. As the amount of hippuric acid cleared by urine increases, high anion gap develops.¹⁷

Other types of renal toxicities due to smelling of the adherents are as follows: proximal tubular disfunction as such in Fanconi syndrome, acute renal insufficiency due to acute toxic tubular necrosis, interstitial nephritis, irreversible renal insufficiency due to distal tubular cell damage, hypercalciuria in chronic users as a result of mobilization of calcium from the bone due to metabolic acidosis and urolithiasis. Reversible hepatorenal insufficiency has been reported in a young patient who was 19-year-old and smelled toluene-based adherents for 3 years.¹⁷

According to a study published in Norway the main route of exposure to toluene is inhalation. Toluene is metabolized mainly in liver and excreted from the body by urine as hippuric acid. The main damage of toluene to the human health is on reproductive, auditory, and central nervous systems. It was observed in radionuclear examination that toluene concentrated in adipose tissue, bone marrow, spinal cord, and white matter of the brain. It was detected to have lower levels in blood, liver, and kidneys. Toluene crosses the placenta. It is metabolized mainly by oxidation. This process takes place over cytochrome P450 system inside the endoplasmic reticulum of liver parenchymal cells. Ninety-nine percent of the biotransformed toluene is oxidized to benzoic acid over benzyl alcohol and benzaldehyde. Moreover, benzoic acid transforms to glycine or glucuronic acid and hippuric acid and benzoyl glucuronide. The main way of excretion from the body for toluene is by the kidneys (as hippuric acid). The other metabolites in the urine are P‐tolylmercaptouric acid and S‐benzylmercaptouric acid. A very small amount is excreted by urine without changing. And an amount of <2% is taken to the intestines by the help of bile and removed by feces.¹⁸

Smelling toluene causes reversible renal damage that presents itself as renal tubular acidosis. Sometimes this damage is irreversible (tubular disfunction and interstitial nephritis).

In this study, kidneys of the rats that are exposed to thinner inhalation for a defined period and dissected at weeks 1, 3, and 5, respectively, were examined histopathologically. The results were compared with the control group rats that were never exposed to thinner and were sacrificed at the end of week 5. Great deals of the articles in the literature were case studies regarding tubular dysfunctions because of thinner exposure.

Presence or absence of granulomas in kidneys, absence of inflammation and if it was present the fact whether it was focal or diffuse were considered. Development of inflammation because of thinner apparently differed between the groups of week 1 and week 5, and this was the beginning of the way leading to renal insufficiency. There was no difference in granuloma development. If the experiment lasted longer, there might have been granuloma development.

In conclusion, the fact that thinner inhalation does cause detectable damage in the kidney of experimental animals has been proven, parallel to the experimental results and reported cases. As inhalation period extends, the damage becomes more prominent.

Nevertheless the fact that the lesions have also been seen in the control group as well, is a little confusing. A possible explanation can be infection with a probable agent found in the same environment and passed over to the other animals. Although the inhalation process was performed under a closed glass cage, the trace amount of thinner vapor that could have mixed with the air shared with control animals when placed back could be the other probable reason for the lesions.

In order to establish better the real damage of thinner upon kidney, there is a need for more clinical and experimental studies performed in more sterilized settings for longer periods.

CONCLUSION

In countries with low socioeconomic conditions, thinner is the drug of abuse for many youngsters, owing to the expense, difficulty to obtain, and illegality of other drugs. It is widely used in industry, and people are exposed to thinner through inhalation, skin contact, or oral intake. This solvent is mainly composed of toluene, which is responsible for the damage to the central nervous system, respiratory tract, renal and hepatobiliary system when it is inhaled.

In this study, we observed the development of inflammation in the kidneys and this lesion became more remarkable as exposure time extended. Development of inflammation because of thinner apparently differed between the groups of week 1 and week 5 and this was the beginning of the way leading to renal insufficiency. There was no difference in granuloma development. If the experiment lasted longer, there might have been granuloma development.

As we established disorders in movement and consciousness in the rats during thinner inhalation, we can say that thinner also has a damaging effect upon the central nervous system.

In order to prevent the use of thinner as a narcotic, society should be informed about its harmful effects and more effective banning measures should be taken. For those who are occupationally exposed to thinner, preventive measure should be taken at work.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.