calea zacatechichi ( also called calea ternifolia or  dream herb  ) is a flowering plant native to central america and has a long tradition of use as a medicinal plant in indigenous cultures . exposure through inhalation ( smoking ) or ingestion ( as tea ) is primarily used to temporarily intensify lucid dreaming . it is also widely consumed to treat problems associated with the gastrointestinal and endocrine systems . it has recently been marketed as a dietary supplement in the management of diabetes due to its ability to induce hypoglycemic effects [ 35 ] although its mechanism(s ) of action remain unclear . the oneirogenic and other biological effects of c. zacatechichi are attributed in part to their flavones and germacrolides components [ 610 ] . however , flavones represent a class of flavonoids that have been shown to carry cytotoxic effects in part through induction of cytochrome p450 enzyme expression [ 1113 ] . in addition , germacrolides are part of the class of sesquiterpene lactones , which can also exhibit negative effects on both prokaryotic and mammalian cells . the cytotoxicity of both flavonoids and sesquiterpene lactones has been exploited for use as therapy against cancer [ 15 , 16 ] . despite clear evidence that at least some of the biologically active components of c. zacatechichi have the potential to be cytotoxic , safety evaluations of whole forms of this herbal supplement are lacking , especially ones that focus on the kidney . the kidneys use a complex transport system to eliminate unwanted chemicals , regulate blood pressure and glucose levels , and maintain a balanced ph . however , as the glomerular filtrate passes through the tubular system , the reabsorption of water and electrolytes by the proximal tubule cells can progressively concentrate chemicals in the lumen that do not get reabsorbed . unfortunately , the proximal tubules can become exposed to toxic concentrations of such chemicals , even when blood concentrations are relatively lower , leaving the kidneys vulnerable to injury . in the case of c. zacatechichi , it is unknown whether any of its components can be nephrotoxic , but given that it is marketed to diabetics , any preexisting diabetic nephropathy marked by glomerular or proximal tubule damage [ 1820 ] could induce further kidney damage . therefore , we focused our research on screening for the potential nephrotoxicity of c. zacatechichi using an in vitro model of human proximal tubule cells . we chose the hk-2 cell line as our human proximal tubule model for its robust performance in many in vitro toxicology studies [ 2125 ] . we compared the effects of exposing hk-2 cells to c. zacatechichi and two control compounds , a known renal toxicant ( cisplatin ) and a known renal protectant ( valproic acid ) , and evaluated their dose - dependent effects on cytotoxicity , mitochondrial injury , and four kidney - specific biomarkers of toxicity [ 2628 ] : ( 1 ) kidney injury molecule-1 ( kim-1 ) , ( 2 ) albumin , ( 3 ) cystatin c , and ( 4 ) 2-microglobulin ( b2 m ) . albumin , cystatin c , and b2 m are indicators of impaired reabsorption by the proximal tubules . in this study , we demonstrate that c. zacatechichi is capable of inducing both cellular and organellar toxicity in proximal tubule cells . voucher samples of c. zacatechichi deposited at the university of mississippi , national center for natural products research ( ncnpr ) ( ncnpr # 2443 ) , were authenticated using macroscopy and microscopy methods by an ncnpr botanist . a methanol - extract of c. zacatechichi was provided in lyophilized form by ncnpr and was stored in the dark at 4c in a vacuum chamber . dried extract of c. zacatechichi compounds were putatively identified by matching exact mass of analytes with components of c. zacatechichi reported in the literature [ 8 , 3033 ] . hk-2 cells were grown , maintained , and treated in a manner similar to that described previously . stock treatment solutions of c. zacatechichi , nephrotoxicant ( positive control ) cis - diamineplatinum(ii ) dichloride ( cisplatin ) ( sigma - aldrich , st . louis , mo ) , and nephroprotectant ( negative control ) valproic acid ( sigma - aldrich ) were made by weighing out their powders , dissolving them in dmso , and diluting this mixture with media for a final dmso stock solution of 0.4% or less . cells were incubated overnight and treated in triplicate for 24 hours at the dose range of 01000  g / ml . treatment - related cytotoxicity was determined using the established celltiter - glo cell viability assay ( promega , madison , wi ) following the manufacturer 's recommendations . the premise of this luminescent assay is that atp production is directly proportional to cell viability , as atp is central to energy required for vital cellular processes . treated cells in black - wall , clear bottom 96-well plates were equilibrated to room temperature for 30 minutes , during which time water in the outer wells was replaced with approximately 100  ul of treatment or media only controls . following that , an equal volume of celltiter - glo working solution was added to each well . plates were placed on an orbital shaker for 2 minutes to induce cell lysis and then incubated for an additional 10 minutes before being read on an omg fluorostar plate reader ( bmg labtech , ortenberg , germany ) to measure the levels of luminescence emitted from each well . quantification of reactive oxygen species ( ros ) was determined using promega 's ros - glo h2o2 luminescence - based detection system and data were normalized to cell viability . following 24  hrs of direct exposure to c. zacatechichi , cells were incubated with h2o2 substrate and detection reagent , as recommended in the manufacturer 's instructions . changes in mitochondrial membrane potential ( mmp ) were evaluated using the ratiometric dye jc-10 ( enzo , farmingdale , ny ) . cells that were directly exposed to c. zacatechichi were stained with 20  um jc-10 ( final concentration ) for 2 hours , washed , and then read by plate reader ( omg fluorostar ) . excitation was set at 485  nm and emission at 520 and 590  nm was measured . culture supernatants from cells treated for 24 hours with c. zacatechichi , cisplatin , and valproic acid at doses of 333 and 111  g / ml were evaluated for levels of biomarkers of kidney toxicity : kidney injury-1 ( kim-1 ) , albumin , cystatin c , and beta-2-microglobulin ( b2 m ) using the human kidney toxicity kits ( bio - rad , hercules , ca ) . following the manufacturer 's protocol , plates were blocked , washed , and incubated with samples , standard solutions detection antibodies , before being given a final wash . microsoft excel and prism ( graphpad , san diego , ca ) were used for calculations and analyses of all data collected . student 's t - tests or 2-way anovas were used to determine whether dose - matched treatment effects were statistically significant at p values less than 0.01 or 0.001 as indicated . lc - high resolution ms found 231 total molecular features in the c. zacatechichi extract . of these , 24 features had exact mass consistent with that of reported components of c. zacatechichi ( figure 1 ) . the major components based on peak volume , calein a , ciliarin , acacetin , and calealactone c , accounted for about 50% of the known compounds and 8% of the total compounds [ 8 , 3033 ] . to investigate the nephrotoxicity of c. zacatechichi , we performed an atp - based cell viability assay on hk-2 cells treated with a 6-dose concentration range from 0 to 1000  g / ml for 24 hours . for comparison , we also treated cells for 24 hours with dose - matched concentrations of the known nephrotoxic compound , cisplatin , and the known nephroprotectant , valproic acid . we found that cisplatin induced a significant reduction in cell viability starting at the ~12  g / ml dose tested ( p < 0.001 ) and caused complete cell death at the maximum dose tested ( figure 2 ) . similarly , significant cytotoxicity of c. zacatechichi was detected starting at 37.0  g / ml ( p < 0.001 ) and still achieved complete cell death by 1000  g / ml . for the range of doses tested , the cytotoxic effect of c. zacatechichi was directly proportional to the treatment dose and we calculated its lethal concentration 50 ( lc50 ) value to be 91.7  g / ml , compared to 13.3  g / ml for cisplatin . by contrast , valproic acid successfully maintained cell viability across the range of tested doses , except for the maximum dose of 1000  g / ml , where cell viability dropped only slightly , as shown in figure 2 . as expected from a nephroprotectant , the calculated lc50 value of valproic acid is quite high at 3866  g / ml , given the plateau shape of its cell viability curve . to begin studying early events of cellular toxicity , we evaluated how the mitochondria of hk-2 cells were affected by 24-hour treatments with c. zacatechichi relative to treatments with cisplatin or valproic acid . we first measured the levels of ros produced in treated cells to indicate the levels of oxidative stress that was created in the intracellular environment of hk-2 cells . using a luminescence assay of ros detection , we found that c. zacatechichi treatment led to increasingly higher levels of ros production in a manner that was directly proportional to the increasing treatment dose ( figure 3(a ) ) . ros production from cells treated with up to 333  g / ml of c. zacatechichi was intermediary between those from the positive- and negative - control treated cells . at the 1000  g / ml dose , however , c. zacatechichi induced ros levels that surpassed those in cisplatin - treated cells ( p < 0.001 ) , as shown in figure 3(a ) . to gain a better understanding of ( 1 ) whether the elevated levels of ros production actually correlated with mitochondrial injury and ( 2 ) to what extent injury took place , we performed a ratiometric assay using jc-10 dye to compare the relative levels of damaged and healthy mitochondria in treated hk-2 cells . compared to the baseline ratio value of about 5 , treatment with c. zacatechichi led to a uniquely sharp increase in the ratio of damaged to healthy mitochondria starting from the 12.3  g / ml testing dose and achieved a maximum ratio value of about 50 when the treatment dose was increased to just 37.0  g / ml ( figure 3(b ) ) . this maximal relative level of mitochondrial damage was statistically significant ( p < 0.001 ) and was well sustained for the remaining higher treatment doses of c. zacatechichi . by contrast , cisplatin induced mitochondrial damage at a much slower rate to achieve a ratio value of ~50 at 333  g / ml . as expected , the mitochondrial injurious effects from valproic acid were minimal over the spectrum of treatment doses . to address whether renal cell function would become compromised after treatment with c. zacatechichi , we evaluated cellular biomarkers that are strong indicators of nephrotoxicity [ 2628 ] . we used a sensitive multiplex approach to simultaneously detect differences in the levels of four fda - qualified biomarkers : kim-1 , albumin , cystatin c , and b2 m . we quantitated the concentrations of these analytes in the culture supernatants of hk-2 cells exposed to 111 or 333  g / ml of c. zacatechichi , cisplatin , valproic acid , or untreated media for 24 hours ( figure 4 ) . in agreement with our findings of cellular and mitochondrial toxicity , we found significantly elevated levels for nearly all of these markers ( p < 0.01 ) in culture supernatants of hk-2 cells treated with c. zacatechichi compared to those treated with valproic acid or left untreated . this trend was also observed at the lower treatment dose of 111  g / ml , even though the actual concentrations of each biomarker were typically 10-fold less than in high - dose treatments , as shown in figure 4 . although c. zacatechichi is not a controlled substance under united states federal law , it has been banned in the state of louisiana as well as in poland on the basis of its mind - altering effects [ 34 , 35 ] . in our study , we used an in vitro human renal proximal tubule cell model to perform several assays that collectively evaluated the nephrotoxicity potential of c. zacatechichi . we used its alcohol extract to best model the tincture dietary supplements marketed in the united states . by comparing its toxicity profile to that of a highly toxic pure compound , cisplatin , and an innocuous pure compound , valproic acid although we identified several of the chemical components of c. zacatechichi , we were focused on evaluating the toxicity of this herbal extract as a whole . in vitro testing not only provides a window into cell - specific effects   but also yields informative data on the mechanism(s ) of toxicity . we chose to use the human renal proximal tubule epithelial cell line , hk-2 , because the proximal tubule plays a critical role in controlling the clearance and reabsorption processes of xenobiotics and their metabolites [ 38 , 39 ] . proximal tubule epithelial cells encounter toxicants that are filtered and are an important component of overall nephrotoxicity that can lead to both acute and chronic kidney damage . the hk-2 cell line is an appropriate choice for establishing a renal cell toxicology profile on c. zacatechichi for two main reasons . first , hk-2 cells are human derived and thus , data generated from this cell line are not confounded by differences between human and other species . second , hk-2 cells closely recapitulate many aspects of the morphological and metabolic phenotype of proximal tubule cells in vivo [ 23 , 41 ] . in our evaluation of cytotoxicity , we found striking similarities between our tested herbal extract and cisplatin at single dose treatment concentrations as low as approximately 37  g / ml in the form of short - term exposure . moreover , it appeared that the mechanism of action of c. zacatechichi 's active ingredients or renal - derived metabolites resembled those of the highly injurious cisplatin ; elevated ros levels and a severe loss of mitochondrial membrane potential were hallmarks of nephrotoxicity shared by these two substances . by contrast , valproic acid showed little or no toxicity potential until the highest dose of 1000  g / ml was tested . the relatively high level of toxicity that was induced by c. zacatechichi within the 24 hours of direct exposure to hk-2 cells is of concern . however , since no data exist on the serum concentrations of c. zacatechichi 's active components , it is unclear how our chosen treatment doses compare to what kidney cells in vivo would be exposed to , especially postmetabolism by the gut and liver . although further studies would be needed to elucidate a more detailed mechanistic analysis of c. zacatechichi 's modes of action , we have found additional evidence of its potential to cause significant renal cell damage . specifically , our panel of indicators of kidney injury showed that not only were these biomarkers elevated , but also the intensity of their elevation approached that measured in our assays following cisplatin treatment . the biomarkers we selected included those that have been qualified by the fda to serve as official biomarkers of nephrotoxicity . they have gained attention recently as they have been shown to be solid correlates of in vivo nephrotoxicity . overall , our findings indicated that the cellular toxicity of c. zacatechichi was capable of producing elevations in all four biomarkers at the high treatment dose , but to a lesser extent than cisplatin . other toxicology studies on c. zacatechichi using in vivo model systems have not specifically evaluated nephrotoxicity endpoints but have still demonstrated its potential to have a range of side effects . in a rat model , for example , extracts of this herb were reported to inhibit edema and neutrophil migration . in a feline model , it caused ataxia , vomiting , and unusual electroencephalogram ( eeg ) recordings . in human volunteers , it resulted in significant increases in respiratory rates and decreases in reaction times . in support of the idea that c. zacatechichi has the potential to cause cell injury , other groups have shown that extracts of this herb or its purified components can exert inhibitory effects on cells using in vitro model systems . for example , a recent toxicology study has shown that c. zacatechichi can inhibit the transcription factor nf - kappab , which is critical to regulating cellular inflammation and other functions [ 45 , 46 ] . a further understanding of c. zacatechichi 's mechanism(s ) of action may be extrapolated from studies on other members of the calea genus . for example , c. platylepis , c. uniflora , and c. serrata have been shown to possess potent antimicrobial , antifungal , and acaricidal activities , respectively [ 4749 ] . in addition , c. pinnatifida was shown to have cytotoxic effects against a wide variety of human cell lines derived from a variety of organ systems , including kidney . moreover , studies on germacrolides , which are common components of most herbs in the calea genus , including c. zacatechichi , have demonstrated the potential for antileishmania effects , inhibition of cellular differentiation , and cytotoxicity against human leukemia cells [ 52 , 53 ] . taken together , c. zacatechichi or its components may pose unwanted health effects , especially if long - term daily doses are taken to control hyperglycemia . our in vitro hk-2 proximal tubule cell model depicted potentially nephrotoxic features of this herb at both the cellular and organellar levels . it would be pertinent to next perform an in vivo investigation of its systemic and organ - specific effects , including those on the other parts of the kidney . highlights . ( i ) in vitro exposure of human kidney cells to calea zacatechichi is cytotoxic . ( iii ) biomarkers of nephrotoxicity are elevated following in vitro exposure to calea zacatechichi .
a recent surge in the use of dietary supplements , including herbal remedies , necessitates investigations into their safety profiles . 
 dream herb ,  calea zacatechichi , has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the us for medicinal purposes , including diabetes treatment . despite the inherent vulnerability of the renal system to xenobiotic toxicity , there is a lack of safety studies on the nephrotoxic potential of this herb . 
 additionally , the high frequency of diabetes - associated kidney disease makes safety screening of c. zacatechichi for safety especially important . 
 we exposed human proximal tubule hk-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of c. zacatechichi relative to control compounds . 
 we evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant . 
 moreover , these findings correlated with significantly elevated levels of nephrotoxicity biomarkers , lending further support for the need to further scrutinize the safety of this herbal dietary supplement .