Dear Maarten. I am concerned that all the information provided in response to your question may be confusing and appear, in some respects, contradictory. So how can the answers be reconciled? A few indisputable facts about STZ might help.

STZ has two intracellular effects accounting for it’s cellular toxicity: DNA alkylation and NAD+/NADH depletion by a PARP dependent mechanism, the latter being suggested to be most important in beta cell toxicity. This matters as it demonstrates that STZ will be toxic to any cell within which it achieves adequate intracellular concentrations.

STZ is taken up into cells by GLUT2, differential expression of GLUT2 by different tissues explaining much (but not all) of the differential tissue toxicity and the well described strain/species differences. It is a very toxic compound so effective use requires a concentration to be found that results in a toxic concentration in beta cells but a non-toxic concentration in the other tissues that it will enter. In our early use of STZ (20 years ago!) we performed autopsies on animals that died and found liver and kidney to be particularly affected, which is not surprising now that we know about GLUT2 mediated uptake. If the dose is too high widespread toxicity will be seen. Unfortunately the safe and effective window is quite narrow. For mice we titrate new batches in 20mg/kg steps. I have attached data from one such experiment. The formatting has scrambled a bit but the X-axis is fasting glucose at day 3 post injection. The other important data is that 40% mortality was seen in the 180mg/kg group and no mortality was seen in any other group. You will see that the dose window is quite small.

This is all fairly basic stuff. What is crucially important is that, just like glucose, STZ exists in α and β anomeric forms. This is not the same as the D/L enantiomers where the whole molecule is a mirror image, but defines the position of a single hydroxyl group: often referred to as up or down! This is important for a couple of reasons. GLUT2 only transports α glucose efficiently and therefore similarly only transports α STZ. As a consequence of this it is also not quite true to say the α anomer is more toxic: it is this anomer that gets in to cells. It is thus also the concentration of the α anomer that is important in determining the toxicity of a particular preparation. The commercially available preparations have a defined composition, typically approximately 75-85% α anomer, in the solid compound.

However, as with all such compounds, the α anomeric and β anomeric forms will equilibrate in solution, so as soon as STZ is dissolved equilibration begins. Much of the degradation of STZ is in fact this equilibration with loss of the α anomer with resulting loss of efficacy. True degradation is slow. Early work describes an effective half life of about 19 minutes, giving some idea of the rate at which equilibration proceeds. The de la Garza-Rodea paper is of course crucial in this regard and describes this chemistry pretty definitively. What this publication does not take into account is the wider aspects of the biology. From memory I think they used the same does of STZ for immediate administration and delayed administration. Given the above these preparations will inevitably and predictably have different efficacy/toxicity. At equilibrium there is a slight excess of β anomer. The rate at which equilibrium is achieved is of course affected by temperature and the buffer used. So aqueous and citrate buffer, pH4-7, solution at room temperature or kept on ice and any time point for injection from 10 minutes to 3 hours are all possible. What will differ is the percentage of the initial α anomer that is still present in solution and therefore the toxicity of the particular prep at that particular time. STZ equilibrates particularly quickly in aqueous solutions at room temperature, much less quickly in citrate buffer at pH 4-4.5 on ice. The problem with the 2-3 hour equilibrated protocol is that this preparation will have approximately half the concentration of ‘active’ α anomer compared to fresh STZ. Hence the lower incidence of diabetes in many peoples hands and the suggestion that multiple doses are needed. Diabetes can be consistently and reliably induced with a single dose if all the above parameters are considered when deciding on the protocol for your particular experiments. Given your logistic constraints I can see that the equilibrated protocol might work best, but do make sure that you work out the correct dose.

I mention this as it is crucially important to recognise these differences as the dose of STZ used by one person may not be right in your model with your logistic constraints.

I should add that the nutritional status of the animal affects the response. This is obvious from the above as α D-glucose will of course compete with α STZ for uptake and the amount of glucose ‘available’ will therefore impact on beta cell toxicity. Administration of carbohydrate post injection has been advocated to counter the hypoglycaemia that can be seen with insulin release from targeted beta cells. This may be true (although we have not found this to be a big problem with correct doses and may relate to overdosing with hepatic toxicity preventing also counter regulatory mechanisms). This post dose carbohydrate may be more important in preventing ongoing toxicity. This was important in our experiments as we were transplanting islets so needed to avoid toxicity to the transplanted tissue. We found transplanted beta cells to be fine at 3 days post injection of STZ. What matters is that this nutritional variable is controlled also in your protocol. I would suggest that the best way is to fast animals overnight as discussed elsewhere on Research Gate at length. This may be a problem for the multiple dose regimens. I am not a fan of post STZ glucose administration as I have not found it to be necessary and it introduces another variable with regard to beta cell killing to be controlled. But this is a personal view.

So in summary it is all about understanding the biology and chemistry and as a result getting the dose of the α anomer right for your model.

Sorry this post is so long! Hope it helps

Dear Maarten van Beek. Induction of drugs to induce diabetes are commonly used soon after dilution to not allow decrease of the effect. I believe it is not advisable you perform an experiment with a prepared streptozotocin some time before, especially if you make the dilution at the time of induction of diabetes in the next experiments. Good luck!

STZ is extremely unstable. We put it into solution immediately prior to injection. Even 10 minutes is a long time. You can end up with variable results using absolutely fresh STZ versus a 10 minute old solution. Also, you should consider using alloxan instead of STZ. In general, we find that it give more consistent results.

Hi,

I would not recommend doing that. STZ degrades 10 min after dilution, you should inject the animal right after you dissolve stz (it happened to me once - I did not get any DM in rats when I dissolved stz within 15 min before the injection). It's better not to store dissolved stz, too, even at -80.

it is also very important to keep the pH of the solvent at 4.5, otherwise stz may not work either.

Best,

Daria

Fresh is always good, I did see a difference in Diabetes induction if the STZ solution is old (like 30 mins or more). I like to give 5 days injections...this will decrease the mortality and I increased the dose day by day (first day 40mg/kg bodg weight) with final dose on day 5 being 50mg/kg body weight. This is in Mice.

For Rats I always gave one time Tail Vein Injections....but from what I have seen Rats are sensitive than mice in Handling STZ, so you have to carefully monitor them (in blood glucose levels, sucrose in water, Insulin Injections) and there is no other choice. Hope this helps

We had problems with STz toxicity a couple of years ago so followed the publication you cite . We get consistent diabetes in rate when preparing STz (6.5mgml in citrate buffer ph 4.5h ) and leaving in fridge for up to 1 hour before ip dosing at 10mlkg. We get consistent hyperglycemia within 48hours (give choice of sucrose in drinking water for 2 days post injection) and rarely have a problem with toxicity. We have been running studies for over 2 years using this protocol ( sigma sources STZ). Good luck.

I meant Rats not rate .

I suspect that the benefits of waiting 2-3 hours were because the initial dose of STZ was too high and 2-3 hours resulted in degradation and reduced efficacy. A lower dose injected immediately would have the same effect and be more predictable. I agree with all above comments. What is important is that you understand these time effects and batch to batch variability. I always advocate some initial dose ranging experiments to work out what works in your hands given the very narrow window of efficacy for STZ. If your local constraints are difficult to overcome I would not absolutely rule out a delay >10 min but would be nervous about waiting 2-3 hours and it would be important to keep the delay constant.

I have used this exact anomer equilibration protocol. I have gotten much more consistent results. I don't want to completely ablate the beta-cells, though. In any case, all my animals receiving the anomer-equilibrated solution thus far have become diabetic (blood glucose >300 mg/dl), and none have died from hypoglycemia due to massive insulin dump into the bloodstream when beta-cells are killed off, or from extreme hyperglycemia due to no beta-cells left. I definitely had encountered those problems using our previous "inject within 15-min" protocol.

I agree with Fred and Lisa, Alloxan would be a better alternative instead of STZ; However, to get more consistent results, you may also utilize the methodology Lisa mentioned with some level of variation as STZ is very much unstable and even refrigerated STZ after one hr of storage time may yield unpredictable results.

Just after my comment, Richard added a very important point and that is as STZ degrades at an exponential rate, the time delay which may not be minimized as such, must be maintained as constant across the board. This will ensure consistent results and eliminate the confounding effect of time lapse. Again, this delay may be in couple of tens of minutes but not couples of hours. You may also do some experimentation to find the relationship in decay#time and the efficacy#dose#time of the agent.

Best of luck in your research.

I worked extensivelya few years ago with STZ to induce diabetes in rats. I made my solutions in cold citrate buffer at pH4.5 as the literature suggests, made up the syringes for ip injection on a per kilogram basis, and then had help to inject the solutions aimed at completion over the next 10-20 minutes. In my >100 rats, I had about 10% failure to develop diabetes, but this was not related to the time after making up the injection and in fact on one ocasion all six in a batch of rats became diabetic despite a 20-30minute gap.

More recently, I repeated the work in pigs, where the injection needed to be iv and also needed to be slow, partly to prevent vomiting which is a problem in the pig with STZ, according to the literature. Out of my 10 pigs, two failed to become diabetic and with one of these, I tried a further three times to provoke the diabetic change, escalating the dose/kg substantially. The last dose did make it vomit, whereas we otherwise avoided this, but it never did become diabetic although the BG rose a little for a few days to just above normal and then reverted. So, I think it likely that for whatever reason, some pigs and probably some rats are resistant to the effects. Our animals were sometimes litter mates but not always, although the breeds for both rats and pigs were consistent and from the same provider.

When I was doing the rat work, I wondered if sometimes we had inadvertently injected into gut lumen and effectively lost the dose, but following the pig experience where the dose was vascular, I am now tempted to think that pancreatic blood supply may differ and be protective in some animals, although I have no evidence for this, other than the observation that in both pig and rat, the pancreas is a branched filmy organ quite unlike the human version and could possibly have non-standard circulation.

You could try dissolve the STZ in sterile 0.9% NaCl instead of citrate buffer, our experience is that it will be more stable (up to 30 min). Otherwise I suggest you switch to Alloxan.

Alloxan is much more unstable in aqueous soln.

I have induced diabetes using STZ from sigma chemicals and used the preparation 1 hr after after preparation so I again prepared the solution and administered the same in another group of rats but no significant difference in results.

Thanks for your help. Really appreciate it!

I was indeed planning to keep the time window constant for all animals. So if I would wait longer because I have no choice due to regulations, then I would fix the timepoint of injection to be the same in all of them. Since there are quite some people having success with longer time intervals I might at least try it out in my pilot study!

For those of you who waited longer than 10 mins. How long did you follow up after injection and monitored diabetes? For my project it is important to have substantial diabetes to induce neuropathy. I might therefore also increase the dose since I wait longer. Thanks!

65mgkg ip (dosed between 30min-1hour post solution preparation ) induces hyperglycaemia within a few days and is maintained for at least 6 weeks.

Mararten: We have standardized ours at 2 h. We collect some animals at 1 week after the last STZ dose, and some animals at 7 weeks (we do multiple low-dose protocol). All of the diabetics have remained diabetic, but not as diabetic as with fresh STZ.

There are couple of points to consider.

1. There is absolutelyno substitute for need of fresh soln of stz or alloxan. I suppose an attempt can be made to standardize the period of time up to which a particular concentration of STZ retains its diabetogenecity. When the soln is not too fresh, its possible to have variations between two trials.

2. If substantial diabetes is what you want, then there ca be no compromise on the dose or freshness of STZ reagent. Alloxan is a better diabetogen, induces heavy hyperglycemia which just keeps on getting worse. But the problem with alloxan is mortality that may follow a very brief period of heavy diabetes.

Isnt it possible that you take required amt of stz in a vial placed in a cold -20 pack, as well as some buffer in a different container? Once you reach the site where animals are housed, you can quickly dissolve and administer.

I added a part of a review manuscript that has been submitted to a journal. Hope that you find your answer.

There is no standard protocol for the preparation, dose, or administration of STZ [1]. Maximum stability of STZ is at pH 4 [2](6). However, despite a general believe that STZ should be dissolved immediately before use [3-10], is unstable at neutral pH [5], and low pH solution is required to maintain its stability [1], it has been reported that STZ solutions with a pH of 7.2 are as stable as those with a pH of 4.5 [1] and STZ solution is relatively stable at pH of 7.4 and 37º C at least up to one hour [11] and pH 6.7-7.8 on ice for 30 min [1]. These observations have questioned the need for low pH, low temperature, and immediate use of STZ solutions [1].

1. Deeds, M.C., et al., Single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models. Lab Anim, 2011. 45(3): p. 131-40.

2. Battell, M.L., et al., Treatment and pharmacological intervantions in streptozotocin diabetes, in Experimental models of diabetes, J.H. McNeil, Editor. 1999, CRC PressI Llc. p. 195-216.

3. Masiello, P., et al., Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes, 1998. 47(2): p. 224-9.

4. Rabbani, S.I., K. Devi, and S. Khanam, Inhibitory effect of glimepiride on nicotinamide-streptozotocin induced nuclear damages and sperm abnormality in diabetic Wistar rats. Indian J Exp Biol, 2009. 47(10): p. 804-10.

5. Junod, A., et al., Diabetogenic action of streptozotocin: relationship of dose to metabolic response. Journal of Clinical Investigation, 1969. 48(11): p. 2129.

6. Pierre, W., et al., Hypoglycemic and hypolipidemic effects of Bersama engleriana leaves in nicotinamide/streptozotocin-induced type 2 diabetic rats. BMC Complement Altern Med, 2012. 12: p. 264.

7. Eizirik, D.L. and R.H. Migliorini, Reduced diabetogenic effect of streptozotocin in rats previously adapted to a high-protein, carbohydrate-free diet. Diabetes, 1984. 33(4): p. 383-8.

8. Pitkin, R.M. and W.A. Reynolds, Diabetogenic effects of streptozotocin in rhesus monkeys. Diabetes, 1970. 19(2): p. 85-90.

9. Fierabracci, V., et al., Oral tungstate treatment improves only transiently alteration of glucose metabolism in a new rat model of type 2 diabetes. Endocrine, 2002. 19(2): p. 177-84.

10. Novelli, M., et al., Metabolic and functional studies on isolated islets in a new rat model of type 2 diabetes. Mol Cell Endocrinol, 2001. 175(1-2): p. 57-66.

11. Lenzen, S., The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia, 2008. 51(2): p. 216-26.

We have also had issues with stability of STZ and variability in mortality/diabetic state induction and decided to move to 30 min-60 min timescale post STZ dissolution in ice cold citrate buffer pH 4.5, wrapping containers in foil and keeping them on ice until injection @ 65 mg/kg i.p. So to account for the time taking to inject I make batches of it up at different time points to account for say batches of 6 animal amounts. We also aliquot the STZ dry powder on arrival and keep these in the freezer before use to minimise freeze thaw of the material. This appears to have resolved most of these issues but also keep a note of the batch numbers you use as we have had the odd 'blip' in either mortality or non-induction of STZ which I am pretty sure is due to the STZ. We also are pretty strict with the animal weight we use 300-400g and supply them with 2% sucrose for the first 24 hours to help them through the hypoglycaemic phase they go through when the beta cells necrose and release insulin .

I would have to agree with Kumar. You have to work it out with a fresh preparation, in case you want make your results consistent and acceptable for publication. We take take ice container to procedure room, and perform injections under a hood.

Dear Maarten. I am concerned that all the information provided in response to your question may be confusing and appear, in some respects, contradictory. So how can the answers be reconciled? A few indisputable facts about STZ might help.

STZ has two intracellular effects accounting for it’s cellular toxicity: DNA alkylation and NAD+/NADH depletion by a PARP dependent mechanism, the latter being suggested to be most important in beta cell toxicity. This matters as it demonstrates that STZ will be toxic to any cell within which it achieves adequate intracellular concentrations.

STZ is taken up into cells by GLUT2, differential expression of GLUT2 by different tissues explaining much (but not all) of the differential tissue toxicity and the well described strain/species differences. It is a very toxic compound so effective use requires a concentration to be found that results in a toxic concentration in beta cells but a non-toxic concentration in the other tissues that it will enter. In our early use of STZ (20 years ago!) we performed autopsies on animals that died and found liver and kidney to be particularly affected, which is not surprising now that we know about GLUT2 mediated uptake. If the dose is too high widespread toxicity will be seen. Unfortunately the safe and effective window is quite narrow. For mice we titrate new batches in 20mg/kg steps. I have attached data from one such experiment. The formatting has scrambled a bit but the X-axis is fasting glucose at day 3 post injection. The other important data is that 40% mortality was seen in the 180mg/kg group and no mortality was seen in any other group. You will see that the dose window is quite small.

This is all fairly basic stuff. What is crucially important is that, just like glucose, STZ exists in α and β anomeric forms. This is not the same as the D/L enantiomers where the whole molecule is a mirror image, but defines the position of a single hydroxyl group: often referred to as up or down! This is important for a couple of reasons. GLUT2 only transports α glucose efficiently and therefore similarly only transports α STZ. As a consequence of this it is also not quite true to say the α anomer is more toxic: it is this anomer that gets in to cells. It is thus also the concentration of the α anomer that is important in determining the toxicity of a particular preparation. The commercially available preparations have a defined composition, typically approximately 75-85% α anomer, in the solid compound.

However, as with all such compounds, the α anomeric and β anomeric forms will equilibrate in solution, so as soon as STZ is dissolved equilibration begins. Much of the degradation of STZ is in fact this equilibration with loss of the α anomer with resulting loss of efficacy. True degradation is slow. Early work describes an effective half life of about 19 minutes, giving some idea of the rate at which equilibration proceeds. The de la Garza-Rodea paper is of course crucial in this regard and describes this chemistry pretty definitively. What this publication does not take into account is the wider aspects of the biology. From memory I think they used the same does of STZ for immediate administration and delayed administration. Given the above these preparations will inevitably and predictably have different efficacy/toxicity. At equilibrium there is a slight excess of β anomer. The rate at which equilibrium is achieved is of course affected by temperature and the buffer used. So aqueous and citrate buffer, pH4-7, solution at room temperature or kept on ice and any time point for injection from 10 minutes to 3 hours are all possible. What will differ is the percentage of the initial α anomer that is still present in solution and therefore the toxicity of the particular prep at that particular time. STZ equilibrates particularly quickly in aqueous solutions at room temperature, much less quickly in citrate buffer at pH 4-4.5 on ice. The problem with the 2-3 hour equilibrated protocol is that this preparation will have approximately half the concentration of ‘active’ α anomer compared to fresh STZ. Hence the lower incidence of diabetes in many peoples hands and the suggestion that multiple doses are needed. Diabetes can be consistently and reliably induced with a single dose if all the above parameters are considered when deciding on the protocol for your particular experiments. Given your logistic constraints I can see that the equilibrated protocol might work best, but do make sure that you work out the correct dose.

I mention this as it is crucially important to recognise these differences as the dose of STZ used by one person may not be right in your model with your logistic constraints.

I should add that the nutritional status of the animal affects the response. This is obvious from the above as α D-glucose will of course compete with α STZ for uptake and the amount of glucose ‘available’ will therefore impact on beta cell toxicity. Administration of carbohydrate post injection has been advocated to counter the hypoglycaemia that can be seen with insulin release from targeted beta cells. This may be true (although we have not found this to be a big problem with correct doses and may relate to overdosing with hepatic toxicity preventing also counter regulatory mechanisms). This post dose carbohydrate may be more important in preventing ongoing toxicity. This was important in our experiments as we were transplanting islets so needed to avoid toxicity to the transplanted tissue. We found transplanted beta cells to be fine at 3 days post injection of STZ. What matters is that this nutritional variable is controlled also in your protocol. I would suggest that the best way is to fast animals overnight as discussed elsewhere on Research Gate at length. This may be a problem for the multiple dose regimens. I am not a fan of post STZ glucose administration as I have not found it to be necessary and it introduces another variable with regard to beta cell killing to be controlled. But this is a personal view.

So in summary it is all about understanding the biology and chemistry and as a result getting the dose of the α anomer right for your model.

Sorry this post is so long! Hope it helps

This will definitely help! Thanks a lot Dr. Smith.

Especially since the wider biological actions of STZ haven't been examined on the long term I think it is wise to say early is better (with the right dose). I will use STZ in order to induce diabetic neuropathy and study the chronic phase and will therefore keep the rats in terms of months. This timespan might allow differential effects of STZ to become more prominent -when more equilibrated- and influence other parameters in my model. Moreover the rats have to stay diabetic throughout the study which favors a selective toxicity on the beta cells, since otherwise I might have to work with higher or multiple doses to keep them diabetic which again could contribute to the differential effects.

I am glad to let you know I made an appointment with the safety officer to discuss possibilities to overcome my logistical challenge.

Again, also thanks to everybody else who contributed!

was just thinking if your issue is you can't use the powder form to make up in your unit so that its fresh, if you have a vaccum pump in your unit why not weigh the STZ in the bottom (usually kept sterile) 50 ml falcon tube part of a steriflip unit in your lab, connect it to another tube containing the citrate buffer so that its sealed and you don't need to open a powder containing tube, then once in the unit attach vaccum pump, hey presto fresh STZ solution....might get round all the issues you are experiencing 

I totally agree with Dr. Smith.

on the effect of xenobiotics on animals, age, time and sex are important factors.The pharmacological effect of STZ on beta-cells to cause diabetes may not be achieved in 30mins.Dose dependency is another factor.