Abstract
Ketamine is one of the common recreational drugs used in rave parties and it is frequently taken with alcohol. In spite of this, the potential toxicity of ketamine in liver and kidney has not been fully documented. In this study, ICR mice were treated for periods of 6, 16 and 28 weeks with 30 mg/kg ketamine injected daily intraperitoneally, and together with alcohol (0.5 ml of 10% alcohol for each mouse) during the last 4 weeks of the treatment periods. Our experimental results showed significant damage in liver, including fatty degeneration of liver cells, fibrosis and increase in liver glutamic oxaloacetic transaminase, proliferative cell nuclear antigen and lactate dehydrogenase after 16 weeks of treatment with ketamine. Hydropic degenerations of the kidney tubules were observed as early as 6 weeks of treatment. Long-term ketamine administration (28 weeks) led to atresia of glomeruli in the kidney. Proteinuria was confirmed in the 67% of the ketamine-treated animals after 28 weeks of treatment. It was apparent that ketamine when taken chronically (16 weeks of treatment and thereafter) affected both liver and kidney definitively. The damages in both liver and kidney of these mice were more severe when the animals were treated with both ketamine and alcohol.
Introduction
Ketamine was discovered in 1960s. It was used as an anaesthetic for both human and animals. 1 Several decades later, ketamine lost its favour largely because of its association with psychic emergence reactions such as hallucination. 1 In the last two decades, it was used mainly (a) in paediatric surgery; (b) in surgery of high-risk patients; (c) in veterinary surgery; (d) for pain relief and analgesia; and (e) for opioid sparring effects in abusers. 1 Clinical usage of ketamine as anaesthetic and for pain relief might be justified, as harmful effects were minimal if not frequently applied. 2,3
Unfortunately, in the last 10 years, ketamine has been used as an abusive agent by the younger generation in some Asian countries, with a high percentage of addiction for long term. 4 This trend has in fact begun to spread to America and Europe. 5 –7 Data from animal models had demonstrated adverse effects in the central nervous system due to the long-term ketamine treatment; these included neuronal apoptosis and alteration in hyperphosphorylated tau formation as well as down regulation of sensory 8,9 and cerebellar activities. 10
Organs other than those of the nervous system might also be affected. For example, long-term ketamine treatment led to inflammation, muscular atrophy and eventually fibrosis of the urinary bladder 11,12 and decreased motility of sperm in rodents. 12 However, ketamine effect on liver and kidney has never been studied. Abusers of ketamine usually consumed ketamine and alcohol together in ‘rave parties’, and damages attributable to ketamine alone or combined with alcohol were unclear. It was reported recently that ketamine and alcohol might potentiate each other’s action and increase apoptosis in the cerebellum of treated mouse. 10 This report is a new study on damages inflicted on the liver and kidney by treatment with ketamine alone or ketamine combined with alcohol.
Methods
Animal studies
Approval for this study had been obtained from the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong and the HKSAR Government (License number: (10-17) in DH/HA&P/8/2/1 Pt.10). A total of 90 one-month-old male ICR mice of 30 g each were used. The animals were randomly allotted into 3 study groups based on the treatment periods of 6, 16 and 28 weeks. Apart from the control, each study group was further divided into two subgroups. One treatment subgroup was treated with ketamine alone and the other with ketamine together with alcohol. Daily intraperitoneal (i.p.) injection of 30 mg/kg ketamine was given to all experimental group mice. The rationale for the dosage was documented in a previous article. 12 For the combined ketamine and ethanol treatment subgroup, 0.5 ml of 10% ethanol was given orally to each mouse every day during the last 4 weeks of the study period. The amount of alcohol administered was based on the fact that (a) wine normally contained 12% of alcohol and (b) an average human of 50 kg weight would consume 500 ml of wine per day. This meant that theoretically an individual of 50 g weight should use 0.5 ml alcohol. Since mouse had a much higher metabolic rate than human, 12 we used 0.5 ml for a 30 g mice. Saline was injected i.p. to the controls of the different study periods. In this experiment, alcohol was only given for 4 weeks as the preliminary result indicated that alcohol–ketamine interaction had drastic effects on organs as early as 2 weeks after combinational usage. These data also indicated that longer-term combinational treatment could lead to a much higher mortality. Thus, for this first study, we decided on a 4-week treatment. We might work on ketamine–alcohol interaction for longer terms in future study. Upon completion of treatments, all animals were killed by cervical dislocation and urine samples were collected directly from the urinary bladder. Livers and kidneys were removed and processed as described below.
Histological studies
The kidneys from mice of different subgroups were divided into midsagittal halves together with the excised livers and were fixed in 4% paraformaldehyde, dehydrated in alcohol, cleared in xylene, embedded in paraffin and sectioned at 5 μm thickness. After sectioning, they were deparaffinized, rehydrated and immersed in Mayer’s haematoxylin for 5 min. After washing with water, the sections were dipped in 0.1% acid water and then immersed in Scott’s tap water for 1 min to develop a blue colour. They were then immersed in 1% eosin for 5–10 min to develop a red colour. The sections were then run through in ascending alcohol series, cleared in xylene and mounted in Permount.
Sirius red study on the liver
Slides of livers from control and experimental groups were stained with Sirius red (Merck Chemicals, Darmstadt, Germany) for 1 h and further washed in 2 changes of acidified water to visualize collagen fibres of connective tissue. After this, the slides were further dehydrated in 3 changes of 100% ethanol, cleared in xylene and mounted in Permount.
Immunostaining for lactate dehydrogenase
Kidney and liver sections were first dewaxed, rehydrated and permeabilized for 10 min with 1× phosphate-buffered saline (PBS) supplemented with 0.1% Triton-X and 0.05% Tween 20, followed by three rinses for 5 min each in 1× PBS. The endogenous peroxidase activity was blocked by 3% hydrogen peroxidase in methanol for 45 min. After 3 more rinses in 1× PBS, nonspecific binding was suppressed with 1.5% normal blocking serum for 30 min. The sections were then incubated with primary antibodies lactate dehydrogenase-A (LDH-A (N-14) 1:200; Santa Cruz Biotechnology®, Inc., Germany; sc-27230) overnight at 4°C. On the following day, sections were rinsed thrice with 1× PBS before incubation with respective diluted biotinylated anti-goat secondary antibodies (1:500; Zymed® Laboratories, Inc., USA; 61-1640) for 2 h and then rinsed again. Subsequently, the sections were incubated with diluted streptavidin-horseradish peroxidase (HRP)-conjugated solution (1:500; InvitrogenTM, USA, 43-4323) for 2 hours and rinsed again. The positive staining was then visualized by 0.05% 3,3′ diaminobenzidine tetrahydrochloride in 1× PBS containing 0.01% hydrogen peroxide (H2O2). Then all sections were dehydrated, cleared and mounted with Permount.
Immunostaining for proliferative cell nuclear antigen
Similar to the steps described for immunostaining of LDH, kidney and liver sections were first dewaxed, rehydrated and permeabilized with 1× PBS, supplemented with 0.1% Triton-X and 0.05% Tween 20 for 10 min, followed by 3 rinses in 1× PBS (5 min each), and subsequent immersion in methanol with 3% hydrogen peroxidase for 45 min to block endogenous peroxidase activity. After rinsing with 1× PBS for 3 times and treatment with 1.5% normal blocking serum for 30 min to suppress nonspecific binding, the sections were incubated with diluted primary antibodies against proliferative cell nuclear antigen (PCNA; 1:1000; Abcam®, USA; AB2916) overnight at 4°C. On the following day, after rinsing thrice with 1× PBS, the sections were incubated with respective diluted biotinylated anti-mouse secondary antibodies (1:1000; Zymed® Laboratories, Inc.; 62-6540) for 2 h and then rinsed again. Subsequently, the sections were incubated with diluted streptavidin-HRP-conjugated solution (1:1000; Invitrogen, 43-4323) for 2 h and rinsed again. The positive staining was then visualized by 0.05% 3,3′-diaminobenzidine tetrahydrochloride in 1× PBS containing 0.01% H2O2. The sections were dehydrated, cleared and mounted with Permount. The stained slides were then examined under microscope with magnification at ×100, and the densities of PCNA-positive nuclei of different subgroups were also compared.
Glutamic oxaloacetic transaminase test for liver function
Glutamic oxaloacetic transaminase (GOT), an enzyme found in large amount in the liver, catalyzes the reversible reaction of amino acids. It was used as an indicator for liver functions of the mice. The GOT kit (C010, Nanjing Jiancheng Bioengineering Institute, China) was used for assay. Ten milligrams of liver sampled from different subgroups were weighed and prepared for 1% diluted tissue solutions with 0.75% saline. The diluted solutions were then centrifuged at 3500 r/min. One hundred microlitres of supernatant was removed and added in 500 μl of substrate solution and incubated at 37°C in a water bath for 30 min as instructed in the manual. Corresponding references were also prepared. At the end of the transamination reaction, pyruvate reacted with newly added dinitrophenylhydrazine (DNPN) to form the hydrazone complex. The mixture was further incubated at 37°C in a water bath for 20 min. Five millilitres of 0.4 mol/L sodium hydroxide solution was added to provide an alkaline medium for the formation of a colour complex, the intensity of which was detected at 505 nm by spectrophotometer (Shimadzu Corporation, Japan). The activity of the enzyme in each sample was then quantified and standardized against the protein contents in the sample.
Protein assay paper test
To detect the presence of protein in urine, screening test strips for rapid determination of protein (Medi-Test Combi 3A®, Germany) were used. The test strip was dipped into 2 μl of fresh urine for approximately 1 s. The test strip was then compared with the reference colour scale after 30 s.
Results
Microscopic study of liver from the 6-week ketamine-treated mice exhibited fatty degeneration of liver cells. Many of these liver cells had active nuclei with prominent nucleoli (Figure 1). By 16 weeks of ketamine treatment, some fibrosis was observed at the tips of liver lobes in ketamine-treated animals (Figure 2(a)); while in the control, the corresponding areas of the livers were relatively clear (Figure 2(b)). By 28 weeks of ketamine treatment, fibrosis spread into the parenchyma of the liver in the ketamine-treated mouse (Figure 2(c)) and the extent of fibrosis was more abundant in the livers of animals in the subgroup treated with ketamine plus alcohol, and with large bundles of collagen fibre reaching the centres of lobules (Figure 2(d)). Very few collagen fibres were observed in the parenchyma of the liver of the control (Figure 2(e)). Immunocytochemistry of PCNA (indicating proliferative nuclei) in the control liver had very few positive sites (Figure 3(a)), but in the 28-week ketamine-treated and ketamine-plus-alcohol-treatedsubgroups, the proliferative nuclei (PCNA-positive) were observed throughout the liver (Figure 3(b)). A semi-quantitative histogram depicting the densities of PCNA-positive nuclei (n = 30 fields of 1500 μm2 sizes from each group with magnification at ×100) was illustrated in Figure 3(c). It was clear from this figure that ketamine plus alcohol treatment induced more proliferation of nuclei in the liver than ketamine treatment alone. The elevation of liver enzyme GOT was also recorded, with the highest level in the ketamine-plus-alcohol-treated subgroup (Figure 4).
Microscopic study showing fatty degeneration in liver cells (arrow). Note also very active nucleoli in the nuclei of these cells (×400).
(a) Fibrosis (arrows) was apparent in the 16-week ketamine-treated liver, particularly around the tips of lobules. Sirius red staining was used for collagen fibres (×50). (b) Control liver of equivalent region showed no fibrosis (×50). (c) Ketamine-treated liver in mouse after 28 weeks of treatment showed fibrosis extended into the liver parenchyma (arrow; ×200). (d) Ketamine plus alcohol-treated liver of 28 weeks (alcohol treatment in the last 4 weeks) in the mouse showing extensive fibrosis (arrow; ×400). (e) Control liver with no fibrosis (×400).
(a) Control liver shows no significant proliferative cell nuclear antigen (PCNA)-positive proliferating nuclei (×100). (b) Increase in number of PCNA-positive proliferating nuclei (arrow) was observed throughout the livers of the 16-week and 28-week ketamine-treated animals, in this case, the 28-week ketamine-treated mouse liver (×200). (c) A histogram showing the number of proliferative nuclei in the liver of 28-week control, ketamine- and ketamine-plus-alcohol-treated subgroups (*p < 0.001).
Quantitative analysis of liver enzyme (glutamic-oxaloacetic transaminase: GOT) in the control, ketamine- and ketamine-plus-alcohol-treated mice after 28 weeks of treatment (*p = 0.048; **p < 0.001; ***p = 0.001).
Immunohistochemical studies of the liver in addition reflected an increase in LDH activity in animals after 6 weeks of ketamine treatment (Figure 5(a)) when compared with the control (Figure 5(b)). LDH activity in the treated group was spotty and mostly in scattered droplets (Figure 5(a)). The most intense activity of LDH was observed in livers from the 28-week ketamine treatment combined with 4-week alcohol subgroup (Figure 5(c)), compared with those treated with ketamine alone (Figure 5(d)). In the cases of ketamine–alcohol interaction, LDH activity was seen inside the whole cytoplasm of cells (Figure 5(c)), while those of ketamine-treated mice were still spotty and scattered droplet-like (Figure 5(d)). An estimation of the number of LDH-positive cells showed that liver after 6 weeks of ketamine treatment had an average of 100 positive cells per 1500 μm2, 130/1500 μm2 after 28 weeks of ketamine treatment, while in the control, it was 5/1500 μm2. In the liver of combined ketamine plus alcohol treatment group, there were about 160 LDH-positive cells per 1500 μm2.
(a) Lactate dehydrogenase (LDH) immunohistochemistry in the liver of the 6-week ketamine-treated mouse showing spotty activities in some cells (×100). (b) No activity of LDH was observed in the control mouse (×100). (c) Immunohistochemistry of lactic acid dehydrogenase in the liver of 28-week ketamine-plus-alcohol-treated (4 weeks) mice. Note high activity inside whole cells (arrow) surrounded by fibrous tissue (T; ×100). (d) Immunohistochemistry of lactic acid dehydrogenase in the liver of 28-week ketamine-treated mouse (arrow). Though many cells had activities, the activity was not as high as those in Figure c and were spotty in the cells (×100).
With regard to the kidney, histological changes in the 6- and 16-week ketamine-treated animals demonstrated hydropic degenerations in many of the kidney tubules (Figure 6(a)). By 28 weeks of ketamine treatment, atresia of glomeruli in the kidney was observed in both ketamine-treated and ketamine plus alcohol-treated animals (Figure 6(b)). A rough estimate showed that the ketamine subgroup had glomerular atresia of less than 10%, while in the ketamine-plus-alcohol-treated subgroup, it was as much as 20%. Empty space originally occupied by glomerulus was seen in the kidneys from the ketamine-plus-alcohol-treated subgroup (Figure 6(c)).
(a) Hydropic degeneration of kidney tubules (arrow) in the 16-week ketamine-treated mouse (×400). (b) Atresia of glomerulus in the 28-week ketamine-treated mouse (×400). (c) Degenerated glomerulus with space (arrow) in the kidney of the 28-week ketamine-plus-alcohol-treated mouse (×400).
Immunocytochemistry of PCNA (proliferating nuclei) showed positive sites in a few of the kidney tubules of the control. Some tubules of the 28-week ketamine-treated mice and most of the tubules from the ketamine-plus-alcohol-treated subgroup (Figures 7(a, b and c)) had the highest number of PCNA-positive sites, followed by the ketamine-treated and then the control. Figure 7(d) showed the semiquantitation of PCNA nuclei per field in the kidney (n = 30 per group, size of the field was 1500 μm2 and magnification of observation was ×100) of the three groups.
(a) Proliferative cell nuclear antigen (PCNA)-positive sites (arrows) in control mouse kidney (×200). (b) Increase of PCNA-positive sites in the kidney of mouse after 28 weeks of ketamine treatment (arrows; ×200). (c) Further increase in PCNA-positive sites (arrows) in the kidney of mouse that had 28 weeks of ketamine plus alcohol (4 weeks) treatment (×200). (d) Histogram of PCNA-positive sites in kidney of control, ketamine-treated and ketamine-plus-alcohol-treated (4 weeks) mice after receiving 28 weeks of ketamine treatment (*p < 0.001).
Proteinuria was observed in 0% of urine samples from the control, 15% in ketamine-treated and 27% in ketamine-plus-alcohol-treated animals in the 6-week treatment period groups. After 16 weeks of treatment, proteinuria was detected in 0% of the control, 40% of the ketamine-treated and 60% in the ketamine-plus-alcohol-treated subgroups. By 28 weeks of treatment, proteinuria was present in 67% of the ketamine-treated animals and 70% of the animals with combined ketamine–alcohol treatment while that of the control remained at 0%.
Discussion
Our results showed definitive pathological and biochemical changes in both the liver and the kidney after long-term ketamine treatment and damages could be aggravated when ketamine was administrated together with alcohol. Preliminary studies in our laboratory showed that when GOT levels in a 28-week ketamine-treated liver were compared with those treated with alcohol alone for 28 weeks, they were equivalent (135 ± 18 karmen units versus 137 ± 15 karmen units), while the GOT level in the liver upon combined treatment of ketamine and alcohol (for only 4 weeks) were higher (185 ± 22 karmen units). Damages of cells in both organs led to cell death and fibrosis as well as physiological derangements. Our liver studies indicated very high activity of LDH after both ketamine and ketamine plus alcohol treatment. Since increase in LDH has been linked to necrosis, 13,14 the mode of cell death was probably by necrosis. Our previous study reported necrotic cells were also in the kidney. 15 This could explain our observation of the absence of apoptosis (unpublished data) by the TUNEL technique on the liver cells after ketamine or ketamine plus alcohol treatment. This result appeared to have differed from our past report that ketamine induced apoptosis in the prefrontal cortices, hippocampi of monkey and mice and that hypertau-positive cells were present in the prefrontal cortices of the monkey and mice. 8,10 In fact, the mode of ketamine- and ketamine-plus-alcohol-induced cell death in the internal organs could differ from organ to organ.
One of our present findings that ketamine up regulates liver enzyme levels agrees with results from studies on the human. 16 Our long-treatment study further indicated that liver damage caused by ketamine (alone or with alcohol) led to cirrhosis. Chan et al. 17 observed expression of multiple forms of P450 rat liver microsomes after 4 days of ketamine treatment at 10–80 mg/kg. When ketamine was given in combination with cocaine or tetrachloromethane, increased oxidative metabolism or even mortality was documented. 17,18 On the other hand, acute injection of high dose of ketamine (70 mg/kg) could afford hepatoprotection mediated by up regulation of haeme-oxygenase 1 and its end product was carbon monoxide 19,20 and down regulation of inducible nitric oxide synthase. 21 There was, however, difference between short duration treatment and long duration treatment. 22
Increase in toxicity upon ketamine–alcohol cotreatment could be detected after only a short period of treatment such as 4 weeks of alcohol in our study. As we have presently demonstrated, the extent of damage could be quite alarming when compared with ketamine treatment alone. This increased toxicity affects not only the liver and kidney but also the central nervous system, including peripheral sensory sites.
23
Working on rodents, we observed an exponential increase in apoptosis and down regulation of blood oxygen images of functional magnetic resonance imaging activities in the cerebellum after combined ketamine and alcohol treatment when compared with those that received ketamine alone. It could be due to the fact that both ketamine and alcohol acted on the N-methyl-
The aim of this study was to evaluate whether long-term treatment of ketamine could lead to damages in liver and kidney, in addition to other organs like the brain and bladder reported earlier.8–12,15 The results clearly revealed cirrhosis of liver as well as glomerular damage and tubular necrosis in the kidney.
Footnotes
Acknowledgements
We are grateful to Prof. Chow PH for her English corrections.
Funding
This work was supported by the Beat Drugs Fund Association, Hong Kong Government (Project Ref. No.: BDF100052).
Declaration of conflicting interests
The authors declared no conflicts of interest.