Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Abstract

The Aboriginal population of Central Australia use endemic Nicotiana spp. to make asmokeless tobacco product known as pituri that they chew/suck for nicotine absorption. Thisthesis describes the relative abundance of nicotine alkaloids amongst Australian Nicotianaspp., with special focus on the molecular characteristics of nicotine to nornicotine conversion.The most popular chewed species, N. gossei, is investigated for nicotine release andcytotoxicity in comparison to similar products to gain insight into potential hazards to pituriusers.To analyse the alkaloids of Nicotiana leaves, a HPLC-UV method was developed to separateand quantify six closely related alkaloids (nicotine, nornicotine, anatabine, anabasine,myosmine, cotinine). A C18 column with a mobile phase of ammonium formate buffer (pH10.5) separated the six alkaloids within 13 min with detection at 260 nm. Linearity, precisionand reproducibility were satisfactory. The limit of quantification was 2.8 and 4.8 µg/mL fornornicotine and nicotine, respectively, and below 2 µg/mL for other alkaloids. This methodquantifies more alkaloids and in less time than previously reported methods.Tobacco alkaloids are responsible in formation of carcinogenic tobacco specific nitrosaminessuch as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK). To quantify NNN and NNK, a fast LC-MS/MS method was developed using a HILICcolumn with a triple quadrupole tandem mass spectrometry. The linearity, accuracy, recoveryand repeatability for this method were satisfactory, with quantification limit of 2.6 and 4.3ng/mL for NNN and NNK, respectively.From 73 donated seedlots, plant growth under controlled environment conditions wassuccessful for 24 of the 26 recognised Australian Nicotiana taxa. The quantification ofalkaloids in leaves indicated nicotine as the abundant alkaloid in 15 taxa but nornicotine in 9.Nornicotine results from demethylation of nicotine, and is associated with negative effects onhealth. A group of cytochrome P450 genes that are mainly expressed during senescence ordrying is involved in this conversion. The conversion loci were amplified in all studied 24 taxa,and sequenced in 6 selected species that contrast in conversion phenotype. Transcriptaccumulation of the responsible loci in fresh versus dried leaves of low or nonconverter N.gossei, N. excelsior and N. benthamiana maintained a steady level or a slight increase, butincreased by 3 fold in cured leaves of the high converter N. goodspeedii, N. velutina and N.cavicola. This indicates the presence of functional loci that are triggered by curing only in high converter species and poses a potential risk for chewers of these species due to their greaterpotential for nornicotine production.The release of nicotine from the leaves of N. gossei was compared to that from a Swedish snus,the CORESTA reference smokeless tobacco (CRP2), and Nicabate chewing gum. A modelbuccal cavity system was developed and three different chewing conditions, performedmanually were tested over 120 minutes: no chewing action, initial chewing and chewing at 15-minute intervals. To simulate the effect of alkaline wood ash, the effect of alkaline pH on therelease of nicotine from N. gossei dry leaves was also evaluated. Samples of the dissolutionmedia were analysed for alkaloids, and the media resulting from chewing every 15 minuteswas quantified for NNN and NNK. The maximum cumulative nicotine release was producedby pressing every 15 minutes. Nicotine release from N. gossei dry leaves was not increasedunder alkaline condition. Nicotine release from smokeless tobacco products is faster and lessdependent on chewing than that from Nicabate gum. N. gossei leaves showed similar releaseprofile to snus, but contained higher nicotine and also released more nornicotine, anatabine,anabasine, and NNN and NNK into the media which can indicate potential health risks.The in vitro toxicity of the aqueous extract from N. gossei leaves on human lung epitheliumcell survival was compared with CRP2 and pure nicotine in an MTS assay. Results indicatedremarkably lower survival of cells treated with extracts from N. gossei leaves and CRP2 thancells treated with nicotine at similar concentrations as present in the extracts. Nicotine in N.gossei leaves and CRP2 was not responsible for their cytotoxicity. Extracts contained othercompounds, including NNN and NNK, which might be responsible for the higher toxicity.In conclusion, Australian Nicotiana species used in pituri vary in their alkaloid compositions,with genetics driving some to contain higher nornicotine. These high-nornicotine species aremore hazardous for chewing as pituri, especially when cured (dried). The nicotine release ratefrom N. gossei dry leaves, which is the main species used in pituri, is similar to Swedish snusand isn’t increased by chewing. The extract from N. gossei dry leaves results in higher toxicityto human lung epithelium cells compared to nicotine at similar concentrations. Altogether,these results indicate the need for further investigation of pituri, its chemicals, and effects onhealth, as pituri remains a popular smokeless tobacco product within some AustralianAboriginal communities.